Protein biomarkers and therapeutic targets for autoimmune and alloimmune diseaes

ABSTRACT

A method for characterizing the risk a subject will develop an autoimmune and/or alloimmune disease following tissue transplant includes obtaining a biological sample from the subject, wherein the subject has received the tissue transplant determining in the biological sample a level of at least one protein selected from Tables 1-4, comparing the measured level of the at least one protein to a control value, and characterizing a subject as at greater risk of developing an autoimmune disease and/or alloimmune disease if the level of at least one protein determined is increased or decreased compared to the control value.

RELATED APPLICATION

This application claims priority from U.S. Provisional Application No.61/182,870, filed Jun. 1, 2009, the subject matter which is incorporatedherein by reference.

BACKGROUND

Autoimmune diseases are generally believed to be caused by the failureof the immune system to discriminate between antigens of foreigninvading organisms (non-self) and tissues native to its own body (self).When this failure to discriminate between self and non-self occurs andthe immune system reacts against self antigens, an autoimmune disordermay arise. Autoimmune diseases, or diseases having an autoimmunecomponent, include rheumatoid arthritis, multiple sclerosis, systemiclupus erythromatosis (SLE), scleroderma, diabetes, inflammatory boweldisease, psoriasis and atherosclerosis. “Alloimmune diseases” arereferred to herein as disorders such as graft versus host disease andtissue transplant rejection, in which an immune response against or byforeign, transplanted tissue can lead to serious complications or befatal. In the treatment of these disorders, it is desired to prevent thebody from reacting against non-self antigens.

Allogeneic hematopoietic stem cell transplantation (SCT) is a curativetherapy for malignant and non malignant conditions. Unfortunately, anumber of complications can occur due to SCT treatment. One majorcomplication is diffuse lung injury, which occurs in 25% to 55% of SCTrecipients and can account for approximately 50% of all SCT relatedmortality. Lung injury following SCT can be infectious or noninfectiousand recent advances in treatment of infectious lung injury has reducedthe incidence of this complication. However, an increase in pulmonarylung injury from non-infectious etiologies has been observed andnon-infectious lung injury poses a significant clinical challenge, as itis associated with significant morbidity and mortality. One type ofnon-infectious lung injury is idiopathic pneumonia syndrome (IPS) whichis defined as widespread alveolar injury following SCT without an activelower respiratory tract infection. It is considered a clinical syndromewith the pathogeneses and diagnostic criteria of this complicationremaining undefined. The incidence of IPS ranges from 5 to 25% with amedian onset of approximately 14 days and an overall day 100 mortalityof 80% despite aggressive treatment therapies.

IPS encompasses a spectrum of clinical presentations and is thought toresult from a diversity of lung insults, including toxic effects ofmyeloablative conditioning, immunologic cell-mediated injury,inflammatory cytokines, and occult pulmonary infections (Fukuda et al.(2003) Blood. 102(8): 2777). Lung biopsies in IPS show diffuse alveolardamage, organizing or acute pneumonia, and interstitial lymphocyticinflammation. The clinical presentation and radiographic findings do notdifferentiate between infectious and idiopathic pneumonia. Ofteninfection needs to be excluded by bronchoalvelor lavage (BAL) or lungbiopsy. Because IPS mimics infectious pneumonia, treatment regimes forIPS include supportive care measures in conjugation with broad-spectrumanti-microbial agents with or without intravenous therapy but responsesare limited and the mortality of patients who develop this diseaseremains high.

IPS continues to cause transplantation-related morbidity and mortalitydespite advances in diagnostic methods for opportunistic infections andrefinements in supportive care. IPS typically occurs early after SCT,therefore advances in transplantation medicine, such as thecharacterization and prevention of IPS could alter the spectrum of lunginjury in patients who have undergone a hematopoietic stem celltransplant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates interaction profiles plots of 3 selected proteins.The x-axis for each plot represents the time point and the y-axis is theintensity after data transformation in a log₂ scale as described in the“Experimental Procedures” section. The solid line represents the medianintensity value for peptide (s) and the dotted line is their estimated95% confidence interval for the median difference. The values for IPSprogressors subjects is indicated with a (P) and the values for controlsubjects is indicated with a (C). Ranking was done by minimum rank ofpeptides. IPI0032311: LBP Lipopolysaccharide-binding protein;IPI00020986: LUM Lumican; IPI00163207: PGLYRP2 Isoform 1 ofN-acetylmuramoyl-L-alanine amidase.

FIG. 2 is a graphical illustration of the distribution of averagelumican peptide intensity (non-log scale) across individual subjects atday 0 for control (C) and IPS (T) in accordance with an example of thepresent invention. Subjects highlight with a star denote IPS subjectswho responded to treatment.

FIG. 3 is a graphical illustration of average lumican peptide intensity(non-log scale) across individual subjects at day 0 in accordance withanother example of the present invention.

FIG. 4 is a graphical illustration of average Mannose-binding protein C(MBL2) intensity (non-log scale) across individual subjects at day 0 inaccordance with another example of the present invention.

FIG. 5 is a graphical illustration of average peptidoglycan recognitionprotein (PGLYRP2) (non-log scale) across individual subjects at day 0 inaccordance with another example of the present invention.

SUMMARY OF THE INVENTION

The present invention relates to the identification and measurement ofproteins whose abundance levels in a biological sample from a subjectcan be used to characterize the risk a subject will develop and/or theprogression of an autoimmune disease and/or alloimmune disease, such asIdiopathic Pneumonia Syndrome (IPS), following tissue transplant.Compared to existing methods of characterizing autoimmune disease and/oralloimmune disease following tissue transplant, the protein expressionprofiles disclosed herein constitute a more robust signature ofautoimmune disease and/or alloimmune disease, and provide a morereliable basis for the characterization of a subject's disease as wellas the selection of appropriate therapeutic regimens.

In general, the invention involves the use of expression profiles of themarker proteins listed in Tables 1, 2, 3 and 4 for characterizing therisk a subject will develop an autoimmune disease and/or alloimmunedisease following tissue transplant.

TABLE 1 Protein Description A1BG Alpha-1B-glycoprotein A2MAlpha-2-macroglobulin AFM Afamin AGT Angiotensinogen AHSGAlpha-2-HS-glycoprotein ALDOB Fructose-bisphosphate aldolase B AMBP AMBPprotein APCS Serum amyloid P-component APOA1 Apolipoprotein A-I APOA2Apolipoprotein A-II APOA4 Apolipoprotein A-IV APOA4 apolipoprotein A-IVprecursor APOB Apolipoprotein B-100 APOC3 Apolipoprotein C-III APODApolipoprotein D APOE Apolipoprotein E APOF apolipoprotein FprecursorAPOH Beta-2-glycoprotein 1 APOL1 Isoform 2 of Apolipoprotein-II APOMApolipoprotein M ATRN Isoform 3 of Attractin AZGP1 alpha-2-glycoprotein1, zinc BTD biotinidase precursor C14orf39 Protein SIX60S1 C1QBcomplement component 1, q subcomponent, Bchain precursor C1QC ComplementClq subcomponent subunit C C1R Complement Clr subcomponent C1RLComplement C1r subcomponent-like protein C3 Complement C3 (Fragment) C4AComplement component 4A C4B complement component 4B preproprotein C4BPAC4b-binding protein alpha chain C4BPB Isoform 2 of C4b-binding proteinbeta chain C5 Complement C5 C7 Complement component C7 C8A Complementcomponent C8 alpha chain C8B Complement component C8 beta chain C8GComplement component C8 gamma chain C9 Complement component C9 CFBIsoform 1 of Complement factor B (Fragment) CFH Isoform 1 of Complementfactor H CFHR2 Isoform Short of Complement factor H-related protein 2CFI Complement factor I CLEC3B Putative uncharacterized proteinDKFZp686HI7246 CP Ceruloplasmin CPB2 Isoform 1 of Carboxypeptidase B2CPN1 Carboxypeptidase N catalytic chain CRP Isoform 1 of C-reactiveprotein F2 Prothrombin (Fragment) FCN3 Isoform 1 of Ficolin-3 FGAIsoform 2 of Fibrinogen alpha chain GC vitamin D-binding proteinprecursor GMPR2 GMPR2 protein GPX3 Glutathione peroxidase 3 GSN Isoform1 of Gelsolin HBB Hemoglobin subunit beta HPX Hemopexin HRGHistidine-rich glycoprotein IGFALS Insulin-like growth factor-bindingprotein complex acid labile chain ITIH1 Inter-alpha-trypsin inhibitorheavy chain HI ITIH2 Inter-alpha-trypsin inhibitor heavy chain HZ ITIH3Isoform 1 of Inter-alpha-trypsin inhibitor heavy chain H3 KNG1 IsoformLMW of Kininogen-1 KRT1 Keratin, type II cytoskeletal 1 LBPlipopolysaccharide-binding protein LOC653879 similar to complementcomponent 3 LRG1 Leucine-rich alpha-2-glycoprotein LUM Lumican MBL2Mannose-binding protein C COMPLEMENT COMPONENT C6 PRECURSORCarboxypeptidase N subunit C1S UNCHARACTERIZED PROTEIN ClS F12,COAGULATION FACTOR XII KLKB1, PLASMA KALLIKREIN PRECURSOR ORM1Alpha-1-acid glycoprotein 1 ORM2 Alpha-1-acid glycoprotein 2 PGLYRP2Isoform 1 of N- acetylmuramoyl-L-alanine amidase PLG Plasminogen PON1Serum paraoxonase/arylesterase 1 PONS Serum paraoxonase/lactonase 3 POS1Vitamin K-dependent protein S RBP4 Retinol binding protein 4, plasmaS100A9 Protein Sl00-A9 SAA1; SAA2 Serum amyloid A protein SAA1; SAA2serum amyloid A2 isoform a SAA4 Serum amyloid A-4 protein SERPINA1isoform 1 of Alpha-1- antitrypsin SERPINA3 Isoform 1 of Alpha-1-antichymotrypsin SERPINA4 Kallistatin SERPINA6 Corticosteroid-bindingglobulin SERPINA7 Thyroxine-binding globulin SERPIND1 Serpin peptidaseinhibitor, clade D (Heparin cofactor), member 1 SERPINF1 Pigmentepithelium-derived factor SERPINF2 SERPINF2 protein SERPING1 Plasmaprotease C1 inhibitor SHBG Isoform 1 of Sex hormone-binding globulinSLC26A6 Anchor protein SLC26A6 Long-chain fatty acid transport protein 6TCP11L1 cDNA FU11386 fis, clone HEMBA1000523, weakly similar to TESTIS-SPECIFIC PROTEIN PBS13 TTR Transthyretin UBTF Isoform UBF2 of Nucleolartranscription factor 1 VTN Vitronectin

TABLE 2 Protein Description A1BG Alpha-1B-glycoprotein AFM Afamin AGTAngiotensinogen AHSG Alpha-2-HS-glycoprotein ALDOB Fructose-bisphosphatealdolase B AMBP AMBP protein APCS Serum amyloid P-component APOA1Apolipoprotein A-I APOA2 Apolipoprotein A-II APOA4 Apolipoprotein A-IVAPOB Apolipoprotein B-100 APOD Apolipoprotein D APOE Apolipoprotein EAPOH Beta-2-glycoprotein 1 APOM Apolipoprotein M ATRN Isoform 3 ofAttractin AZGP1 zinc alpha-2-glycoprotein 1 C14orf39 Protein SIX6OS1C1QB complement component 1, q subcomponent, B chain precursor C1QCComplement C1q subcomponent subunit C C1R Complement C1r subcomponent C3Complement C3 (Fragment) C4A Complement component 4A C4BPA C4b-bindingprotein alpha chain C5 Complement C5 C7 Complement component C7 C8AComplement component C8 alpha chain C9 Complement component C9 CFHIsoform 1 of Complement factor H CFHR1 Complement factor H-relatedprotein 1 CFHR2 Isoform Short of Complement factor H-related protein 2CFI Complement factor I CLEC3B Putative uncharacterized proteinDKFZp686H17246 CLU clusterin isoform 1 CP Ceruloplasmin CRP Isoform 1 ofC-reactive protein F10 Coagulation factor X F2 Prothrombin (Fragment)FCGBP IgGFc-binding protein FGA Isoform 2 of Fibrinogen alpha chain GCvitamin D-binding protein precursor GMPR2 GMPR2 protein GPX3 Glutathioneperoxidase 3 GSN Isoform 1 of Gelsolin HPX Hemopexin HRG Histidine-richglycoprotein ITIH1 Inter-alpha-trypsin inhibitor heavy chain H1 ITIH2Inter-alpha-trypsin inhibitor heavy chain H2 ITH3 Isoform 1 ofInter-alpha-trypsin inhibitor heavy chain H3 KNG1 Isoform LMW ofKininogen-1 KRT1 Keratin, type II cytoskeletal 1 KRT10 Keratin, type Icytoskeletal 10 LBP Lipopolysaccharide-binding protein LRG1 Leucine-richalpha-2-glycoprotein LUM Lumican ORM1 Alpha-1-acid glycoprotein 1 ORM2Alpha-1-acid glycoprotein 2 PGLYRP2 Isoform 1 of N-acetylmuramoyl-L-alanine amidase PLG Plasminogen PLTP 45 kDa protein PON1 Serumparaoxonase/arylesterase 1 S100A9 Protein S100-A9 SAA1; SAA2 Serumamyloid A protein SAA1; SAA2 Serum amyloid A2 isoform a SAA4 Serumamyloid A-4 protein SERPINA1 Isoform 1 of Alpha-1-antitrypsin SERPINA3Isoform 1 of Alpha-1- antichymotrypsin SERPINA4 Kallistatin SERPINA7Thyroxine-binding globulin SERPIND1 Serpin peptidase inhibitor, clade DHeparin cofactor member 1 SERPINF1 Pigment epithelium-derived factorSERPINF2 SERPINF2 protein SERPING1 Plasma protease C1 inhibitor SHBGIsoform 1 of Sex hormone-binding globulin SLC26A6 Anchor protein UBTFIsoform UBF2 of Nucleolar transcription factor 1 VTN Vitronectin

TABLE 3 Protein Description F2 Prothrombin (Fragment) C5 Complement C5CFH Isoform 1 of Complement factor H ITIH3 Isoform 1 ofInter-alpha-trypsin inhibitor heavy chain H3 APOA4 Apolipoprotein A-IVAPOB Apolipoprotein B-100 FCGBP IgGFc-binding protein FGA Isoform 2 ofFibrinogen alpha chain KNG1 Isoform LMW of Kininogen-1 C1QC ComplementC1q subcomponent subunit C C9 Complement component C9 SERPINA7Thyroxine-binding globulin CP Ceruloplasmin APCS Serum amyloidP-component C4BPA C4b-binding protein alpha chain F10 Coagulation factorX CFI Complement factor I GPX3 Glutathione peroxidase 3 SLC26A6 Anchorprotein C1R Complement C1r subcomponent SERPING1 Plasma protease C1inhibitor KRT10 Keratin, type I cytoskeletal 10 AMBP AMBP protein C1QBcomplement component 1, q subcomponent, B chain precursor SAA1; SAA2Serum amyloid A protein SERPINF1 Isoform 1 of Alpha-1-antitrypsin CLUclusterin isoform 1 ORM2 Alpha-1-acid glycoprotein 2 SERPINF1 Pigmentepithelium-derived factor PLTP 45 kDa protein ALDOBFructose-bisphosphate aldolase B APOE Apolipoprotein E LRG1 Leucine-richalpha-2-glycoprotein LBP Lipopolysaccharide-binding protein AZGP1 zincalpha-2-glycoprotein 1 CRHR2 Isoform Short of Complement factorH-related protein 2 GMPR2 GMPR2 protein ORM1 Alpha-1-acid glycoprotein 1SAA1; SAA2 serum amyloid A2 isoform a C14orf39 Protein SIX6OS1 SERPINA3Isoform 1 of Alpha-1- antichymotrypsin AGT Angiotensinogen CRP Isoform 1of C-reactive protein CRHR1 Complement factor H-related protein 1 KRT1Keratin, type II cytoskeletal 1

TABLE 4 Protein Description VTN Vitronectin ATRN Isoform 3 of AttractinSHBG Isoform 1 of Sex hormone-binding globulin AHSGAlpha-2-HS-glycoprotein APOM Apolipoprotein M SERPINA4 Kallistatin GSNIsoform 1 of Gelsolin AFM Afamin ITIH2 Inter-alpha-trypsin inhibitorheavy chain H2 CLEC3B Putative uncharacterized protein DKFZp686H17246C8A Complement component C8 alpha chain APOA2 Apolipoprotein A-II HRGHistidine-rich glycoprotein PGLYRP2 Isoform 1 of N-acetylmuramoyl-L-alanine amidase ITIH1 Inter-alpha-trypsin inhibitor heavy chain H1UBTF Isoform UBF2 of Nucleolar transcription factor 1 APOHBeta-2-glycoprotein 1 LUM Lumican APOA1 Apolipoprotein A-I A1BGAlpha-1B-glycoprotein PLG Plasminogen SAA4 Serum amyloid A-4 proteinAPOD Apolipoprotein D HPX Hemopexin S100A9 Protein S100-A9 GC vitaminD-binding protein precursor SERPINF2 SERPINF2 protein PON1 Serumparaoxonase/arylesterase 1 SERPIND1 Serpin peptidase inhibitor, clade DHeparin cofactor member 1 C4A Complement component 4A C7 Complementcomponent C7 C3 Complement C3 (Fragment)

Therefore, in one aspect of the present invention, a method ofcharacterizing the risk a subject will develop an autoimmune diseaseand/or alloimmune disease following tissue transplant is provided. Themethod includes the steps of: (1) obtaining a biological sample from asubject following tissue transplant; (2) determining, in the biologicalsample, a level of at least one protein selected from the groupconsisting of proteins presented in Table 1 (i.e.,Alpha-1B-glycoprotein, Alpha-2-macroglobulin, Afamin, Angiotensinogen,Alpha-2-HS-glycoprotein, Fructose-biphosphate aldolase B, AMBP protein,Serum amyloid P-component, Apolipoprotein A-I, Apolipoprotein A-II,Apolipoprotein A-IV, Apolipoprotein precursor, Apolipoprotein B-100,Apolipoprotein C-III, Apolipoprotein D, Apolipoprotein E, ApolipoproteinF precursor, Beta-2-glycoprotein 1, Isoform 2 of Apolipoprotein-L1,Apolipoprotein M, Isoform 3 of Attractin, alpha-2-glycoprotein 1 (zincbinding), biotimidase precursor, Protein SIX6OS1, complement component 1(q subcomponent, B chain precursor), Complement C1q subcomponent subunitC, Complement C1r subcomponent, Complement C1r subcomponent-likeprotein, Complement C3 (Fragment), Complement component 4A, Complementcomponent 4B preprotein, C4b-binding protein alpha chain, Isoform 2 ofC4b-binding protein beta chain, Complement C5, Complement component C7,Complement component C8 alpha chain, Complement component C8 beta chain,Complement component C8 gamma chain, Complement component C9, Isoform 1of Complement factor B (fragment), Isoform 1 of Complement factor 1-1,Isoform Short of Complement factor H-related protein 2, Complementfactor 1, DKFZp686H17246, Ceruloplasmin, Isoform 1 of CarboxypeptidaseB2, Carboxypeptidase N catalytic chain, Isoform 1 of C-reactive protein,Prothrombin (fragment), Isoform 1 of Ficolin-3, Isoform 2 of Fibrinogenalpha chain, vitamin D-binding protein precursor, GMPR2 protein,Glutathione peroxidase 3, Isoform 1 of Gelsolin, Hemoglobin subunitbeta, Hemopexin, Histidine-rich glycoprotein, Insulin-like growthfactor-binding protein complex acid labile chain, Inter-alpha-trypsininhibitor heavy chain H1, Inter-alpha-trypsin inhibitor heavy chain H2,Isoform 1 of Inter-alpha-trypsin inhibitor heavy chain H3, Isoform LMWof Kininogen-1, Type II cytoskeletal 1 Keratin,Lipopolysaccharide-binding protein, LOC653879 similar to complementcomponent 3, Leucine-rich alpha-2-glycoprotein, Lumican, Mannose-bindingprotein C, Complement component C6 precursor, Carboxypeptidase Nsubunit, Protein CIS, Coagulation factor XII, Plasma KallikreinPrecursor, Alpha-1-acid glycoprotein 1, Alpha-1-acid glycoprotein 2,Isoform 1 of N-acetylmuramoyl-L-alanine amidase, Plasminogen, Serumparaoxonase/arylesterase 1, Serum paraxonase/lactonase 3, VitaminK-dependent protein S, Retinol binding protein 4 (plasma), ProteinS100-A9, Serum amyloid A protein, Serum amyloid A2 isoform a, Serumamyloid A-4 protein, Isoform 1 of Alpha-1-antitrypsin, Isoform 1 ofAlpha-1-antichymotrypsin, Kallistatin, Corticosteroid-binding globulin,Thyroxine-binding globulin, Serpin peptidase inhibitor (Glade D (Heparincofactor), member 1), Pigment epithelium-derived factor, SERPINF2protein, Plasma protease C1 inhibitor, Isoform 1 of Sex hormone-bindingglobulin, Anchor protein, Long-Chain fatty acid transport protein 6,TCP11L1, Transthyretin, Isoform UBF2 of Nucleolar transcription factor1, Vitronectin, analogs and fragments thereof; (3) comparing themeasured level of the at least one protein to a control value; and (4)characterizing a subject as at greater risk of developing an autoimmunedisease and/or alloimmune disease if the level of at least one proteindetermined is increased or decreased compared to the control value.

In some aspects, the biological sample includes a sample of blood,plasma, serum, or bronchoalveolar lavage (BAL) fluid. In some aspects,the tissue transplant includes allogeneic hematopoietic stem celltransplantation.

In some aspects of the present invention, the at least one protein isselected from the group consisting of proteins presented in Table 2(i.e., Vitronectin, Isoform 3 of Attractin, Isoform 1 of Sexhormone-binding globulin, Alpha-2-HS-glycoprotein, Apolipoprotein M,Kallistatin, Isoform 1 of Gelsolin, Afamin, Inter-alpha-trypsininhibitor heavy chain H2, Putative uncharacterized proteinDKFZp686H17246, Complement component C8 alpha chain, ApolipoproteinA-II, Histidine-rich glycoprotein, PGLYRP1, PGLYRP2 Isoform 1 ofN-acetylmuramoyl-L-alanine amidase, Inter-alpha-trypsin inhibitor heavychain H1, Isoform UBF2 of Nucleolar transcription factor 1,Beta-2-glycoprotein 1, Lumican, Apolipoprotein A-I,Alpha-1B-glycoprotein, Plasminogen, Serum amyloid A-4 protein,Apolipoprotein D, Hemopexin, Protein S100-A9, vitamin D-binding proteinprecursor, SERPINF2 protein, Serum paraoxonase/arylesterase 1, Serpinpeptidase inhibitor Glade D (Heparin cofactor) member 1, Complementcomponent 4A, Complement component C7, Complement C3 (Fragment),Prothrombin (Fragment), Complement C5, Isoform 1 of Complement factor H,Isoform 1 of Inter-alpha-trypsin inhibitor heavy chain H3,Apolipoprotein A-IV, Apolipoprotein B-100, IgGFc-binding protein,Isoform 2 of Fibrinogen alpha chain, Isoform LMW of Kininogen-1,Complement C1q subcomponent subunit C, Complement component C9,Thyroxine-binding globulin, Ceruloplasmin, Serum amyloid P-component,C4b-binding protein alpha chain, Coagulation factor X, Complement factorI, Glutathione peroxidase 3, Anchor protein, Complement C1rsubcomponent, Plasma protease C1 inhibitor, Keratin type I cytoskeletal10, AMBP protein, complement component 1 q subcomponent B chainprecursor, Serum amyloid A protein, Isoform 1 of Alpha-1-antitrypsin,clusterin isoform 1, Alpha-1-acid glycoprotein 2, Pigmentepithelium-derived factor, 45 kDa protein, Fructose-bisphosphatealdolase B, Apolipoprotein E, Leucine-rich alpha-2-glycoprotein,Lipopolysaccharide-binding protein, zinc alpha-2-glycoprotein 1, IsoformShort of Complement factor H-related protein 2, GMPR2 protein,Alpha-1-acid glycoprotein 1, serum amyloid A2 isoform a, ProteinSIX6OS1, Isoform 1 of Alpha-1-antichymotrypsin, Angiotensinogen, Isoform1 of C-reactive protein, Complement factor H-related protein 1, andKeratin type II cytoskeletal 1).

In some aspects of the present invention, the at least one protein isselected from the group consisting of proteins presented in Table 3(i.e., Prothrombin (Fragment), Complement C5, Isoform 1 of Complementfactor H, Isoform 1 of Inter-alpha-trypsin inhibitor heavy chain H3,Apolipoprotein A-IV, Apolipoprotein B-100, IgGFc-binding protein,Isoform 2 of Fibrinogen alpha chain, Isoform LMW of Kininogen-1,Complement C1q subcomponent subunit C, Complement component C9,Thyroxine-binding globulin, Ceruloplasmin, Serum amyloid P-component,C4b-binding protein alpha chain, Coagulation factor X, Complement factorI, Glutathione peroxidase 3, Anchor protein, Complement C1rsubcomponent, Plasma protease C1 inhibitor, Keratin type I cytoskeletal10, AMBP protein, complement component 1 q subcomponent B chainprecursor, Serum amyloid A protein, Isoform 1 of Alpha-1-antitrypsin,clusterin isoform 1, Alpha-1-acid glycoprotein 2, Pigmentepithelium-derived factor, 45 kDa protein, Fructose-bisphosphatealdolase B, Apolipoprotein E, Leucine-rich alpha-2-glycoprotein,Lipopolysaccharide-binding protein, zinc alpha-2-glycoprotein 1, IsoformShort of Complement factor H-related protein 2, PGLYRP1, GMPR2 protein,Alpha-1-acid glycoprotein 1, serum amyloid A2 isoform a, ProteinSIX6OS1, Isoform 1 of Alpha-1-antichymotrypsin, Angiotensinogen, Isoform1 of C-reactive protein, Complement factor H-related protein 1, andKeratin type II cytoskeletal 1).

In some aspects of the present invention, the at least one protein isselected from the group consisting of proteins presented in Table 4(i.e., Vitronectin, Isoform 3 of Attractin, Isoform 1 of Sexhormone-binding globulin, Alpha-2-HS-glycoprotein, Apolipoprotein M,Kallistatin, Isoform 1 of Gelsolin, Afamin, Inter-alpha-trypsininhibitor heavy chain H2, Putative uncharacterized proteinDKFZp686H17246, Complement component C8 alpha chain, ApolipoproteinA-II, Histidine-rich glycoprotein, PGLYRP2 Isoform 1 ofN-acetylmuramoyl-L-alanine amidase, Inter-alpha-trypsin inhibitor heavychain H1, Isoform UBF2 of Nucleolar transcription factor 1,Beta-2-glycoprotein 1, Lumican, Apolipoprotein A-I,Alpha-1B-glycoprotein, Plasminogen, Serum amyloid A-4 protein,Apolipoprotein D, Hemopexin, Protein S100-A9, vitamin D-binding proteinprecursor, SERPINF2 protein, Serum paraoxonase/arylesterase 1, Serpinpeptidase inhibitor Glade D (Heparin cofactor) member 1, Complementcomponent 4A, Complement component C7, and Complement C3 (Fragment)).

In some aspects of the present invention, the at least one protein isselected from the group consisting of LPS binding protein,Mannose-binding protein-C, PGLYRP2, atrractin, and lumican.

In some aspects of the present invention, the method can further includegenerating an expression profile based on the determined level of atleast two proteins, comparing the expression profile to a controlexpression profile, and characterizing the subject as having greaterrisk of developing GVHD if the expression profile compared to thecontrol expression profile is substantially different. In some aspectsan increase or decrease of at least 5%, or at least 20%, in determinedlevel of the at least one protein compared to the control valuecharacterizes the subject at greater risk of developing GVHD. In someaspects, the GVHD includes idiopathic pneumonia syndrome (IPS). In someaspects, the IPS is a subtype of IPS syndrome that is responsive totreatment by a TNF-α inhibitor.

In yet another aspect of the present invention, a method ofcharacterizing the progression of IPS in a subject following allogeneichematopoietic stem cell transplantation is provided. The method includesthe steps of (1) obtaining a biological sample from a subject, whereinthe subject has received the allogeneic hematopoietic stem celltransplantation; (2) determining, in the biological sample, the level ofone or more of proteins selected from the group consisting of theproteins presented in Tables 1, 2, 3, 4, and analogs and fragmentsthereof; (3) comparing the measured level of the at least one protein toa control value; and (4) characterizing the IPS as progressing if thelevel of at least one protein determined is increased or decreasedcompared to the control value.

In certain aspects of the present invention, the at least one protein isselected from the group consisting of LPS binding protein,Mannose-binding protein-C, PGLYRP2, attractin and lumican. In someaspects, the biological sample includes a sample of blood, plasma,serum, or bronchoalveolar lavage (BAL) fluid.

In some aspects of the present invention, the method can further includegenerating an expression profile based on the determined level of atleast two proteins, comparing the expression profile to a controlexpression profile, and characterizing the IPS in a subject asprogressing if the expression profile compared to the control expressionprofile is substantially different. In some aspects an increase ordecrease of at least 5%, or at least 20%, in determined level of the atleast one protein compared to the control value characterizes the IPS inthe subject as progressing. In some aspects, the IPS is a subtype ofidiopathic pneumonia syndrome that is responsive to treatment by a TNF-αinhibitor.

In still another aspect, the present invention provides a method forcharacterizing the efficacy of a TNF-α inhibitor in treating anautoimmune disease and/or alloimmune disease in a subject followingtissue transplant. The method includes the steps of: (1) obtaining abiological sample from a subject, wherein the subject has received the atissue transplant; (2) determining, in the biological sample, a level ofone or more of proteins selected from the group consisting of theproteins presented in Tables 1, 2, 3, 4, and analogs and fragmentsthereof; (3) comparing the measured level of the at least one protein toa control value; and (4) characterizing the TNF-α inhibitor as beingmore effective in treating the autoimmune disease and/or alloimmunedisease when administered to the subject if the level of at least oneprotein determined is increased or decreased compared to the controlvalue.

In certain aspects of the present invention, the at least one protein isselected from the group consisting of LPS binding protein,Mannose-binding protein-C, PGLYRP2, attractin and lumican. In someaspects, the biological sample includes a sample of blood, plasma,serum, or bronchoalveolar lavage (BAL) fluid.

In some aspects of the present invention, the autoimmune disease and/oralloimmune disease is graft versus host disease (GVHD). In otheraspects, the GVHD is IPS. In still other aspects, the IPS is a subtypeof IPS syndrome that is responsive to treatment by a TNF-α inhibitor.

In a further aspect, the method can include generating an expressionprofile based on the determined level of at least two proteins andcomparing the expression profile to a control expression profile,wherein a substantial difference in the expression profile compared tothe control expression profile characterizes the TNF-α inhibitor asbeing more effective in treating IPS when administered to the subject.In some aspects an increase or decrease of at least 5%, or at least 20%,in determined level of the at least one protein compared to the controlvalue characterizes the TNF-α inhibitor as being more effective intreating IPS when administered to the subject.

In still yet another aspect of the invention, a method of treating asubject having or at elevated risk of idiopathic pneumonia syndrome(IPS) following allogeneic hematopoietic stem cell transplantation isprovided. The method includes the steps of: (1) obtaining a biologicalsample from a subject, wherein the subject has received the allogeneichematopoietic stem cell transplantation; (2) determining, in thebiological sample, a level of one or more of proteins selected from thegroup consisting of the proteins presented in Tables 1, 2, 3, 4, andanalogs and fragments thereof; (3) comparing the measured level of theat least one protein to a control value; and (4) administering a TNF-αinhibitor to the subject to treat IPS if the level of at least oneprotein determined is increased or decreased compared to the controlvalue.

In certain aspects of the present invention, the at least one protein isselected from the group consisting of LPS binding protein,Mannose-binding protein-C, PGLYRP2, attractin, and lumican. In someaspects, the biological sample includes a sample of blood, plasma,serum, or bronchoalveolar lavage (BAL) fluid.

In some aspects of the present invention, the method can further includegenerating an expression profile based on the determined level of atleast two proteins and comparing the expression profile to a controlexpression profile, and administering a TNF-α inhibitor to the subjectto treat IPS if there is a substantial difference in the expressionprofile compared to the control expression profile. In some aspects, asubject is administered a TNF-α inhibitor if the determined level of theat least one protein compared to the control increases or decreases atleast 5%, or at least 20%. In some aspects, the TNF-α inhibitor includesetanercept.

DETAILED DESCRIPTION

Methods involving conventional molecular biology techniques aredescribed herein. Such techniques are generally known in the art and aredescribed in detail in methodology treatises, such as Current Protocolsin Molecular Biology, ed. Ausubel et al., Greene Publishing andWiley-Interscience, New York, 1992 (with periodic updates). Unlessotherwise defined, all technical terms used herein have the same meaningas commonly understood by one of ordinary skill in the art to which thepresent invention pertains. Commonly understood definitions of molecularbiology terms can be found in, for example, Rieger et al., Glossary ofGenetics: Classical and Molecular, 5th Edition, Springer-Verlag: NewYork, 1991, and Lewin, Genes V, Oxford University Press: New York, 1994.The definitions provided herein are to facilitate understanding ofcertain terms used frequently herein and are not meant to limit thescope of the present invention.

The term “subject” and “individual” are used herein interchangeably.They refer to a human or another mammal that can be afflicted with anautoimmune disease or alloimmune disease, such as idiopathic pneumoniasyndrome, but may or may not have the disease. In many embodiments, thesubject is a human being.

As used herein, the terms “treatment,” “treating,” and the like, referto obtaining a desired pharmacologic and/or physiologic effect. Theeffect may be prophylactic in terms of completely or partiallypreventing a disease or symptom thereof and/or may be therapeutic interms of a partial or complete cure for a disease and/or adverse affectattributable to the disease. “Treatment,” as used herein, covers anytreatment of a disease in a mammal, particularly in a human, andincludes: (a) preventing the disease from occurring in a subject whichmay be predisposed to the disease or at risk of acquiring the diseasebut has not yet been diagnosed as having it; (b) inhibiting the disease,i.e., arresting its development; and (c) relieving the disease, i.e.,causing regression of the disease.

The term “biological sample” is used herein in its broadest sense. Abiological sample may be obtained from a subject (e.g., a human) or fromcomponents (e.g., tissues) of a subject. The sample may be of anybiological tissue or fluid with which the proteins described herein maybe assayed. Frequently, the sample will be a “clinical sample”, i.e., asample derived from a patient. Such samples include, but are not limitedto, bodily fluids, which may or may not contain cells, e.g., blood,blood plasma, serum, bronchoalveolar lavage (BAL) fluid, tissue or fineneedle biopsy samples; and archival samples with known diagnosis,treatment and/or outcome history. Biological samples may also includesections of tissues such as frozen sections taken from histologicalpurposes. The term biological sample also encompasses any materialderived by processing the biological sample. Derived materials include,but are not limited to, cells (or their progeny) isolated from thesample, proteins or nucleic acid molecules extracted from the sample.Processing of the biological sample may involve one or more of,filtration, distillation, extraction, concentration, inactivation ofinterfering components, addition of reagents, and the like.

The terms “normal” and “healthy” are used herein interchangeably. In thepresent context, they can refer to an individual or group of individualswho have not shown any symptoms of an autoimmune disease and/oralloimmune disease following a tissue transplant, and have not beendiagnosed with an autoimmune disease and/or an alloimmune disease, suchas idiopathic pneumonia syndrome (IPS). In certain embodiments, normalindividuals have similar sex, age, body mass index as compared with theindividual from which the sample to be tested was obtained. The term“normal” is also used herein to qualify a sample isolated from anindividual who has not had a tissue transplant.

In the context of the present invention, the term “control sample”refers to one or more biological samples isolated from an individual orgroup of individuals that are normal (i.e., healthy). A control samplecan also refer to a biological sample isolated from a patient or groupof patients prior to tissue transplantation (e.g., a stem celltransplantation). The term “control sample” (or “control”) can alsorefer to the compilation of data derived from samples of one or moreindividuals classified as normal, or one or more individuals diagnosedwith IPS, one or more individuals likely to respond well to TNF-αinhibitor treatment, or one or more individuals having undergonetreatment for IPS.

As used herein, the term “differentially expressed protein” refers to aprotein or polypeptide whose abundance level in a biological sample isdifferent (e.g., increased or decreased) in a subject (or a populationof subjects) afflicted with a GVHD, such as IPS, relative to a controlvalue. The term also encompasses a protein whose level is different insubject afflicted with different subtypes of the disease (e.g., thoselikely to be responsive to treatment by a TNF-α inhibitor). Differentialexpression includes quantitative, as well as qualitative, differences inthe temporal or cellular expression pattern of the biomarker. Asdescribed in greater details below, a differentially expressed protein,alone or in combination with other differentially expressed proteins, isuseful in a variety of different applications in subject and diseasecharacterization, therapeutic, drug development and related areas. Theexpression patterns of the differentially expressed proteins disclosedherein can be described as a fingerprint or a signature of an IPS, IPSsubtype and IPS progression. They can be used as a point of reference tocompare and characterize unknown samples and samples for which furtherinformation is sought.

The term “decreased level” as used herein, refers to a decrease in theabundance level of one or more of the proteins described herein of atleast 5% or more. For example, 5%, 10%, 20%, 30%, 40%, or 50%, 60%, 70%,80%, 90% or more, or a decrease of greater than 1-fold, 2-fold, 3-fold,4-fold, 5-fold, 10-fold, 50-fold, 100-fold or more as measured by one ormore methods described herein. The term “increased level” as usedherein, refers to an increase in the abundance one or more of theproteins described herein of at least 5% or more. For example, 5%, 10%,20%, 30%, 40%, or 50%, 60%, 70%, 80%, 90% or more, or an increase ofgreater than 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 50-fold,100-fold or more as measured by one or more methods, such as a methoddescribed herein.

The terms “protein”, “polypeptide”, and “peptide” are used hereininterchangeably, and refer to amino acid sequences of a variety oflengths, either in their neutral (uncharged) forms or as salts, andeither unmodified or modified by glycosylation, side chain oxidation, orphosphorylation. In certain embodiments, the amino acid sequence is thefull-length native protein. In other embodiments, the amino acidsequence is a smaller fragment of the full-length protein. In stillother embodiments, the amino acid sequence is modified by additionalsubstituents attached to the amino acid side chains, such as glycosylunits, lipids, or inorganic ions such as phosphates, as well asmodifications relating to chemical conversion of the chains, such asoxidation of sulfhydryl groups. Thus, the term “protein” (or itsequivalent terms) is intended to include the amino acid sequence of thefull-length native protein, subject to those modifications that do notchange its specific properties. In particular, the term “protein”encompasses protein isoforms, i.e., variants that are encoded by thesame gene, but that differ in their pI or MW, or both. Such isoforms candiffer in their amino acid sequence (e.g., as a result of alternativesplicing or limited proteolysis), or in the alternative, may arise fromdifferential post-translational modification (e.g., glycosylation,acylation, phosphorylation). In certain embodiments, proteins refer tothose proteins whose expression profile was found to characterize anautoimmune disorder, such as IPS, and/or the likelihood or risk of asubject having a GVHD, such as IPS, or a sub-type of IPS that isresponsive to treatment by a TNF-α inhibitor.

The term “protein analog”, as used herein, refers to a protein thatpossesses a similar or identical function as the full-length nativeprotein but need not necessarily comprise an amino acid sequence that issimilar or identical to the amino acid sequence of the protein, orpossesses a structure that is similar or identical to that of theprotein. Preferably, in the context of the present invention, a proteinanalog has an amino acid sequence that is at least 30% (more preferably,at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95% or at least 99%) identical to theamino acid sequence of the full-length native protein.

The term “protein fragment”, as used herein, refers to a proteincomprising an amino acid sequence of at least 4 amino acid residues(preferably, at least 10 amino acid residues, at least 15 amino acidresidues, at least 20 amino acid residues, at least 25 amino acidresidues, at least 40 amino acid residues, at least 50 amino acidresidues, at least 60 amino acid residues, at least 70 amino acidresidues, at least 80 amino acid residues, at least 90 amino acidresidues, at least 100 amino acid residues, at least 125 amino acidresidues, at least 150 amino acid residues, at least 175 amino acidresidues, at least 200 amino acid residues, or at least 250 amino acidresidues) of the amino acid sequence of a second protein. The fragmentof a marker protein may or may not possess a functional activity of thefull-length native protein.

The terms “labeled”, “labeled with a detectable agent” and “labeled witha detectable moiety” are used herein interchangeably. These terms areused to specify that an entity (e.g., a probe) can be visualized, forexample, following binding to another entity (e.g., a polynucleotide orprotein). Preferably, the detectable agent or moiety is selected suchthat it generates a signal which can be measured and whose intensity isrelated to the amount of bound entity. In array-based methods, thedetectable agent or moiety is also preferably selected such that itgenerates a localized signal, thereby allowing spatial resolution of thesignal from each spot on the array. Methods for labeling proteins arewell-known in the art. Labeled proteins can be prepared by incorporationof or conjugation to a label, that is directly or indirectly detectableby spectroscopic, photochemical, biochemical, immunochemical,electrical, optical, or chemical means. Suitable detectable agentsinclude, but are not limited to, various ligands, radionuclides,fluorescent dyes, chemiluminescent agents, microparticles, enzymes,calorimetric labels, magnetic labels, and haptens. Detectable moietiescan also be biological molecules such as molecular beacons and aptamerbeacons.

The term “IPS expression profile map” refers to a presentation ofexpression levels of a set of proteins in a particular status of IPS(e.g., low risk of subject developing disease, high risk of subjectdeveloping disease, IPS progression, and/or IPS subtype likely torespond positively to TNF-α inhibitor treatment). The map may bepresented as a graphical representation (e.g., on paper or a computerscreen), a physical representation (e.g., a gel or array) or a digitalrepresentation stored in a computer-readable medium. Each mapcorresponds to a particular status of the disease (e.g., low risk ofsubject developing disease, high risk of subject developing disease, IPSprogression, and/or IPS subtype likely to respond positively to TNF-αinhibitor treatment) or to a sample taken from the patient previously,and thus provides a template for comparison to a patient sample. Incertain embodiments, maps are generated from a plurality of samplesobtained from a significant number of control subjects). Maps may beestablished for individuals with matched age, sex and body mass index.

As used herein, the term “IPS subtype that is responsive to treatment bya TNF-α inhibitor” refers to an IPS subtype susceptible to treatmentthrough the administration of a TNF-α inhibitor. In the context of thepresent invention, “responsive to treatment by a TNF-α inhibitor” mayinclude, for example: modulation of the level of at least one of theproteins described herein; and/or to delay or prevent the onset of IPS;and/or to slow down or stop the progression, aggravation, ordeterioration of the symptoms of IPS; and/or to bring about ameliorationof the symptoms of IPS, and/or to cure IPS.

The term “mass spectrometry” or “MS” as used herein refer to methods offiltering, detecting, and measuring ions based on their mass-to-chargeratio, or “m/z.” In general, one or more molecules of interest areionized, and the ions are subsequently introduced into a massspectrographic instrument where, due to a combination of magnetic andelectric fields, the ions follow a path in space that is dependent uponmass (“m”) and charge (“z”).

As used herein, the term “ionization” refers to the process ofgenerating an analyte ion having a net electrical charge equal to one ormore electron units. Negative ions are those having a net negativecharge of one or more electron units, while positive ions are thosehaving a net positive charge of one or more electron units.

The present invention relates to the identification and use of proteinsas systemic non-invasive (e.g., plasma) markers to characterize the riska subject will develop an autoimmune disease and/or alloimmune disease,such as graft versus host disease, after a tissue transplant. Thepresent invention also relates to improved methods and strategies forthe characterization of idiopathic pneumonia (IPS), and sub-typing ofIPS. The invention also relates to therapeutic treatments for IPS aswell as methods of drug discovery.

The present invention is based in part on the discovery that theabundance levels of specific proteins are significantly modulated insubjects with IPS progression after receiving a tissue transplantcompared to control values. It has also been found that changes in theabundance of proteins responsible for regulation of the immune response,such as LPS binding protein, Mannose-binding protein-C, PGLYRP2 protein,attractin, and lumican are significant predictors of the risk a subjectwill develop a GVHD, such as IPS, following tissue transplant.

The present invention provides the identity of a set of proteinsindicative of IPS identified using high-throughput proteomicstechnology. The protein markers indicative of IPS are listed in Tables1, 2, 3 and 4. The inventors analyzed samples of blood plasma obtainedfrom subjects who had received a tissue transplant and who wereafflicted with IPS. The inventors then compared the protein expressionlevel from these samples to samples obtained from subjects who hadreceived a tissue transplant but were not afflicted by IPS. It was foundthat the proteins listed in Tables 1, 2, 3 and 4 can be used todiscriminate between tissue transplant recipients likely to develop IPSand tissue transplant recipients who are not likely to develop IPS asearly as day 0 post-tissue transplant even when signs of non-infectiousdiffuse lung injury related to IPS would not be visible.

It was also found that the proteins listed in Tables 1, 2, 3 and 4 canbe used to discriminate between an IPS subtype which is responsive totreatment through the administration of a TNF-α inhibitor and IPS thatis not likely to be responsive to TNF-α inhibitor treatment. The presentinvention describes specific proteins and assays in biological samplesto detect the identified proteins.

Extensive research in animal models supports non-infectious lung injury,e.g., idiopathic pneumonia syndrome (IPS), as an immune mediated attackthat includes elements of the adaptive and innate immune system. Withoutbeing bound by theory, it is thought that graft-versus-host disease(GVHD) and/or autoimmune phenomena are responsible for the developmentof IPS in subjects who have received a tissue transplant. The earlypost-tissue transplant phase is characterized by the presence ofinflammatory cytokines whose net effect is to promote lymphocyte influxinto lungs with minimal fibrosis that leads to an acute form ofgraft-versus-host reaction-mediated pulmonary tissue damage. Gradualchanges over time in leukocyte influx and activation lead todysregulated wound repair mechanisms resulting from the shift in thebalance of cytokines that promote fibrosis. Thus, it is believed thatcytokine-modulated immunological mechanisms, which occur during theacute and chronic phases after tissue transplantation lead to thedevelopment of the progressive, inflammatory, and fibrotic lung diseasetypical of idiopathic pneumonia syndrome.

As discussed above, GVHD may be responsible for the development of IPSin subjects who have received a tissue transplant. Thus, it is alsocontemplated by the present invention that changes in the abundance ofthe identified protein markers described herein characterize thesubject's risk of developing a GVHD.

Therefore, one aspect of the present invention provides a method forcharacterizing the risk a subject will develop an autoimmune diseaseand/or alloimmune disease, such as GVHD, following tissue transplant.The method includes the steps of: (1) obtaining a biological sample froma subject, wherein the subject has received a tissue transplant; (2)determining, in the biological sample, a level of at least one proteinselected from the group consisting of proteins presented in Tables 1, 2,3, 4, analogs and fragments thereof; (3) comparing the measured level ofthe at least one protein to a control value; and (4) characterizing asubject as at greater risk of developing an autoimmune disease and/or analloimmune disease, such as GVHD, if the level of at least one proteindetermined is increased or decreased compared to the control value.

An autoimmune disease and/or alloimmune disease in a subject can includebut is not limited to idiopathic pneumonia syndrome, rheumatoidarthritis, refractory arthritis, Crohn's disease, psoriasis, psoriaticarthritis, ankylosing sondylitits, idiopathic inflammatory boweldisease, and juvenile idiopathic arthritis.

The control value can be determined from samples obtained from a healthyindividual (or a group of healthy individuals), from an individual (orgroup of individuals) prior to tissue transplantation, from anindividual (or group of individuals) afflicted with IPS, and/or from anindividual (or group of individuals) afflicted with a specific subtypeof the disease (e.g., a subtype of IPS responsive to TNF-αt inhibitortreatment). In some aspects, the control expression levels of thebiomarkers of interest are preferably determined from a significantnumber of individuals, and an average or mean is obtained.

A tissue transplant, as contemplated by the present invention is used inits broadest meaning and refers to both autologous and allogeneic tissuetransplants. In some aspects of the invention, a tissue transplant foruse in the present methods may include, but is not limited totransplantation of bone marrow, blood, stem cells, brain, heart, lung,cornea, fetal tissue, kidney liver, skin and islets of Langerhans. Incertain embodiments, the tissue transplant includes a hematopoietic stemcell transplant.

The methods of the present invention may be further used to characterizethe risk a subject will develop IPS following a hematopoietic stem celltransplant. Therefore, in another aspect of the present invention, amethod of characterizing the risk a subject will develop IPS following ahematopoietic stem cell transplant is provided. The method includes thesteps of: (1) obtaining a biological sample from a subject, wherein thesubject has received the allogeneic hematopoietic stem celltransplantation; (2) determining, in the biological sample, the level ofone or more of proteins selected from the group consisting of theproteins presented in Tables 1, 2, 3, 4, and analogs and fragmentsthereof; (3) comparing the measured level of the at least one protein toa control value; and (4) characterizing a subject as at greater risk ofdeveloping IPS if the level of at least one protein determined isincreased or decreased compared to the control value.

As shown in the Examples below, it has been discovered in subject'sdiagnosed with IPS, that the abundance level of certain proteins willcontinue to increase or decrease over time compared to a control value.Therefore, it is further contemplated by the present invention that anincrease or decrease in the abundance level of proteins found to besignificantly modulated in subjects with IPS progression can bedetermined in order to characterize the progression of IPS in a subject.For example, a sample taken from a subject following a stem celltransplant which includes a significantly increased level of at leastone protein compared to a sample taken from the subject previously, cancharacterize the IPS in the subject as progressing. On the other hand, asample taken from a subject following a stem cell transplant may includea significantly decreased level of at least one protein compared to asample taken from the subject previously. In this case, the decreasedabundance level may characterize the IPS in the subject as notprogressing, and even regressing.

Thus in accordance with another aspect of the present invention, amethod of characterizing the progression of IPS in a subject isprovided. The method includes the steps of (1) obtaining a biologicalsample from a subject, wherein the subject has received the allogeneichematopoietic stem cell transplantation; (2) determining, in thebiological sample, the level of one or more of proteins selected fromthe group consisting of the proteins presented in Tables 1, 2, 3, 4, andanalogs and fragments thereof; (3) comparing the measured level of theat least one protein to a control value; and (4) characterizing the IPSas progressing if the level of at least one protein determined isincreased or decreased compared to the control value.

In some aspects of the invention, information on abundance levels ofproteins in a biological sample obtained from individuals afflicted witha GVHD, IPS or a particular subtype of the disease (e.g., IPS that isresponsive to TNF-α inhibitor treatment) may be grouped to form aspecific expression profile map. In one example, an IPS expressionprofile map results from the study of a large number of individuals withthe same disease sub-type. In some embodiments, an IPS expressionprofile map is established using samples from individuals with matchedage, sex, and body index. Each expression profile map provides atemplate for comparison to protein expression patterns generated fromunknown biological samples. Expression profile maps may be presented asa graphical representation (e.g., on paper or a computer screen), aphysical representation (e.g., a gel or array) or a digitalrepresentation stored in a computer-readable medium. In certain aspectsof the invention, the level of at least one protein in the biologicalsample under investigation is determined and compared to at least oneexpression profile map for IPS, as described above.

The methods of the invention may be applied to the study of any type ofbiological samples allowing one or more inventive protein biomarkers tobe assayed. Examples of biological samples include, but are not limitedto, urine, blood, and blood products (e.g., blood plasma). In aparticular aspect of the present invention, the biological sample isblood plasma obtained from the subject.

The biological samples used in the practice of the inventive methods maybe fresh or frozen samples collected from a subject, or archival sampleswith known diagnosis, treatment and/or outcome history. Biologicalsamples may be collected by any non-invasive means, such as, forexample, by drawing blood from a subject, or using fine needleaspiration or needle biopsy. Alternatively, biological samples may becollected by an invasive method, including, for example, surgicalbiopsy. In certain aspects, the inventive methods are performed on thebiological sample itself without or with limited processing of thesample.

In still other embodiments, the inventive methods are performed on aprotein extract prepared from the biological sample. Preferably, theprotein extract contains the total protein content. However, the methodsmay also be performed on extracts containing one or more of: membraneproteins, nuclear proteins, and cytosolic proteins. Methods of proteinextraction are well known in the art (see, for example “ProteinMethods”, D. M. Bollag et al., 2nd Ed., 1996, Wiley-Liss; “ProteinPurification Methods: A Practical ApprIPSch”, E. L. Harris and S. Angal(Eds.), 1989; “Protein Purification Techniques: A Practical Approach”,S. Roe, 2nd Ed., 2001, Oxford University Press; “Principles andReactions o/Protein Extraction, Purification, and Characterization”, H.Ahmed, 2005, CRC Press: Boca Raton, Fla.). Numerous different andversatile kits can be used to extract proteins from bodily fluids andtissues, and are commercially available from, for example, BioRadLaboratories (Hercules, Calif.), BD Biosciences Clontech (Mountain View,Calif.), Chemicon International, Inc. (Temecula, Calif.), Calbiochem(San Diego, Calif.), Pierce Biotechnology (Rockford, Ill.), andInvitrogen Corp. (Carlsbad, Calif.). User Guides that describe in greatdetail the protocol to be followed are usually included in all thesekits. Sensitivity, processing time and costs may be different from onekit to another. One of ordinary skill in the art can easily select thekits most appropriate for a particular situation. After the proteinextract has been obtained, the protein concentration of the extract ispreferably standardized to a value being the same as that of the controlsample in order to allow signals of the protein markers to bequantitated. Such standardization can be made using photometric orspectrometric methods or gel electrophoresis.

The methods of the present invention generally involve the determinationof the abundance levels of a plurality (i.e., one or more, e.g., atleast 2, at least 3, at least 4, at least 5, at least 6, at least 7, atleast 8, at least 9, at least 10 or more) of proteins in a biologicalsample obtained from a subject. Determination of protein levels in thepractice of the inventive methods may be performed by any suitablemethod (see, for example, E. Harlow and A. Lane, “Antibodies: ALaboratories Manual”, 1988, Cold Spring Harbor Laboratory: Cold SpringHarbor, N.Y.).

In general, protein levels are determined by contacting a biologicalsample isolated from a subject with binding agents for one or more ofthe protein markers listed in Table 1; determining, in the sample, thelevels of proteins that bind to the binding agents; and comparing thelevels of proteins in the sample with the levels of proteins in acontrol sample. As used herein, the term “binding agent” refers to anentity such as a protein or antibody that specifically binds to aninventive protein marker. An entity “specifically binds” to a protein ifit reacts/interacts at a detectable level with the protein but does notreact/interact detectably with peptides containing unrelated sequencesor sequences of different proteins.

In certain aspects of the invention, the binding agent is a peptidecomponent, a protein that comprises a protein sequence of a proteinmarker, a peptide variant thereof, or a non-peptide mimetic of such asequence.

In other aspects, the binding agent is an antibody specific for aprotein marker of the invention. Antibodies for use in the methods ofthe present invention include monoclonal and polyclonal antibodies,immunologically active fragments (e.g., Fab or (Fab)₂ fragments),antibody heavy chains, humanized antibodies, antibody light chains, andchimeric antibodies. Antibodies, including monoclonal and polyclonalantibodies, fragments and chimeras, may be prepared using methods knownin the art (see, for example, R. G. Mage and E. Lamoyi, in “MonoclonalAntibody Production Techniques and Applications”, 1987, Marcel Dekker,Inc.: New York, pp. 79-97; G. Kohler and C. Milstein, Nature, 1975, 256:495-497; D. Kozbor et al., J. Immunol. Methods, 1985, 81: 31-42; and R.J. Cote et al., Proc. Natl. Acad. Sci. 1983, 80: 2026-203; R. A. Lerner,Nature, 1982, 299: 593-596; A. C. Nairn et al., Nature, 1982, 299:734-736; A. J. Czernik et al., Methods Enzymol. 1991, 201: 264-283; A.J. Czernik et al., Neuromethods: Regulatory Protein Modification:Techniques & Protocols, 1997, 30: 219-250; A. J. Czemik et al.,NeuroNeuroprotocols, 1995, 6: 56-61; H. Zhang et al., J. Biol. Chem.2002, 277: 39379-39387; S. L. Morrison et al., Proc. Natl. Acad. Sci.,1984, 81: 6851-6855; M. S, Neuberger et al., Nature, 1984, 312: 604-608;S. Takeda et al., Nature, 1985, 314: 452-454). Antibodies to be used inthe methods of the invention can be purified by methods well known inthe art (see, for example, S. A. Minden, “Monoclonal AntibodyPurification”, 1996, IBC Biomedical Library Series: Southbridge, Mass.).For example, antibodies can be affinity purified by passage over acolumn to which a protein marker or fragment thereof is bound. The boundantibodies can then be eluted from the column using a buffer with a highsalt concentration. Instead of being prepared, antibodies to be used inthe methods of the present invention may be obtained from scientific orcommercial sources.

In certain embodiments, the binding agent is directly or indirectlylabeled with a detectable moiety. The role of a detectable agent is tofacilitate the detection step of the diagnostic method by allowingvisualization of the complex formed by binding of the binding agent tothe protein marker (or analog or fragment thereof). Preferably, thedetectable agent is selected such that it generates a signal which canbe measured and whose intensity is related (preferably proportional) tothe amount of protein marker present in the sample being analyzed.Methods for labeling biological molecules such as proteins andantibodies are well-known in the art (see, for example, “AffinityTechniques. Enzyme Purification. Part B”, Methods in Enzymol., 1974,Vol. 34, W. B. Jakoby and M. Wilneck (Eds.), Academic Press: New York,N.Y.; and M. Wilchek and E. A. Bayer, Anal. Biochem., 1988, 171: 1-32).

Any of a wide variety of detectable agents can be used in the practiceof the present invention. Suitable detectable agents include, but arenot limited to: various ligands, radionuclides, fluorescent dyes,chemiluminescent agents, microparticles (such as, for example, quantumdots, nanocrystals, phosphors and the like), enzymes (such as, forexample, those used in an ELISA, i.e., horseradish peroxidase,beta-galactosidase, luciferase, alkaline phosphatase), colorimetriclabels, magnetic labels, and biotin, dioxigenin or other haptens andproteins for which antisera or monoclonal antibodies are available.

In certain aspects, the binding agents (e.g., antibodies) may beimmobilized on a carrier or support (e.g., a bead, a magnetic particle,a latex particle, a microtiter plate well, a cuvette, or other reactionvessel). Examples of suitable carrier or support materials includeagarose, cellulose, nitrocellulose, dextran, Sephadex, Sepharose,liposomes, carboxymethyl cellulose, polyacrylamides, polystyrene,gabbros, filter paper, magnetite, ion-exchange resin, plastic film,plastic tube, glass, polyamine-methyl vinylether-maleic acid copolymer,amino acid copolymer, ethylene-maleic acid copolymer, nylon, silk, andthe like. Binding agents may be indirectly immobilized using secondbinding agents specific for the first binding agents (e.g., mouseantibodies specific for the protein markers may be immobilized usingsheep anti-mouse IgG Fc fragment specific antibody coated on the carrieror support).

Protein levels in the methods of the present invention may be determinedusing immunoassays. Examples of such assays are radioimmunoassays,enzyme immunoassays (e.g., ELISA), immunofluorescenceimmunoprecipitation, latex agglutination, hemagglutination, andhistochemical tests, which are conventional methods well-known in theart. As will be appreciated by one skilled in the art, the immunoassaymay be competitive or noncompetitive. Methods of detection andquantification of the signal generated by the complex formed by bindingof the binding agent with the protein marker will depend on the natureof the assay and of the detectable moiety (e.g., fluorescent moiety).

Alternatively, the protein levels may be determined using massspectrometry based methods or image (including use of labeled ligand)based methods known in the art for the detection of proteins. Othersuitable methods include proteomics-based methods. Proteomics, whichstudies the global changes of protein expression in a sample, typicallyincludes the following steps: (1) separation of individual proteins in asample by electrophoresis (1-D PAGE), (2) identification of individualproteins recovered from the gel (e.g., by mass spectrometry orN-terminal sequencing), and (3) analysis of the data usingbioinformatics.

In some aspects, one or more proteins can be separated by liquidchromatography (LC) and then conveniently detected and quantified bymass spectrometry (MS). Liquid chromatography removes impurities and maybe used to concentrate the proteins for detection. Traditional LC relieson chemical interactions between sample components and a stationaryphase such as a column packing. Laminar flow of the sample, mixed with amobile phase, through the column is the basis for separation of thecomponents of interest. The skilled artisan understands that separationin such columns is a partition process.

In various embodiments, one or more of the purification and/or analysissteps can be performed in an automated fashion. By careful selection ofvalves and connector plumbing, two or more chromatography columns can beconnected as needed such that material is passed from one to the nextwithout the need for any manual steps. In certain embodiments, theselection of valves and plumbing is controlled by a computerpre-programmed to perform the necessary steps. The chromatography systemcan also be connected in-line to the detector system, e.g., an MSsystem. Thus, an operator may place a tray of purified samples in anautosampler, and the remaining operations are performed under computercontrol, resulting in purification and analysis of all samples selected.In one embodiment, a diverter valve is placed in-line between the LCcolumn and the interface with the MS. The diverter valve directs the LCeffluent into a waste container until slightly prior to the timeexpected peak retention). This prevents the solvent front and otherimpurities from being passed into the MS device.

As used here, “in-line” refers to a configuration in which the LC andthe ionization/injection device for the first MS quadropole arefunctionally connected in order that the LC effluent passes directlyinto the first MS device. “In-line” configurations may include aselector valve such that the effluent from two or more LC columns may bedirected individually into the MS device and, optionally, to a wastecontainer. Such a configuration is useful for a high throughput systemand reduces the analysis time required for a large number of samples.High throughput systems may be designed in which an autosamplerinitiates LC purifications on the two or more LC columns at staggeredintervals. In this way, the purified protein of interest peak is elutedfrom each LC column at a known interval. In certain embodiments, thepurified protein peaks eluting from the two or more LC columns aredirected into the MS device in rapid succession, but with sufficienttemporal separation that individual measurements are not compromised.Such a high throughput system reduces the amount of “idle-time” for MSdetection attributable to the LC procedure, which typically requiresmore time than the MS analysis.

By contrast, “off-line” refers to a configuration that requires manualintervention to transfer the LC effluent to the MS device. Typically,the LC effluent is captured by a fractionator and must be manuallyloaded into a MS device or into an autos ampler for subsequent MSdetection. Off-line configurations are useful, but less desirablebecause of the increased time required to process large numbers ofsamples.

In an aspect of the invention, the mass-to-charge ratio can bedetermined using a quadrupole analyzer. For example, in a “quadrupole”or “quadrupole ion trap” instrument, ions in an oscillating radiofrequency field experience a force proportional to the DC potentialapplied between electrodes, the amplitude of the RF signal, and m/z. Thevoltage and amplitude can be selected so that only ions having aparticular m/z travel the length of the quadrupole, while all other ionsare deflected. Thus, quadrupole instruments can act as both a “massfilter” and as a “mass detector” for the ions injected into theinstrument.

“Tandem mass spectrometry” or “MS/MS” can be employed to enhance theresolution of the MS technique. In tandem mass spectrometry, a parention generated from a molecule of interest may be filtered in an MSinstrument, and the parent ion subsequently fragmented to yield one ormore daughter ions that are then analyzed (detected and/or quantified)in a second MS procedure.

Collision-induced dissociation (“CID”) is often used to generate thedaughter ions for further detection. In CID, parent ions gain energythrough collisions with an inert gas, such as argon, and subsequentlyfragmented by a process referred to as “unimolecular decomposition.”Sufficient energy must be deposited in the parent ion so that certainbonds within the ion can be broken due to increased vibrational energy.

By careful selection of parent ions using the first MS procedure, onlyions produced by certain analytes of interest are passed to thefragmentation chamber to generate the daughter ions. Because both theparent and daughter ions are produced in a reproducible fashion under agiven set of ionization/fragmentation conditions, the MS/MS techniquecan provide an extremely powerful analytical tool. For example, thecombination of filtration/fragmentation can be used to eliminateinterfering substances, and can be particularly useful in complexsamples, such as biological samples.

The mass spectrometer typically provides the user with an ion scan; thatis, the relative abundance of each m/z over a given range (e.g., 10 to1200 amu). The results of an analyte assay, that is, a mass spectrum,can be related to the amount of the analyte in the original sample bynumerous methods known in the art. For example, given that sampling andanalysis parameters are carefully controlled, the relative abundance ofa given ion can be compared to a table that converts that relativeabundance to an absolute amount of the original molecule.

Alternatively, molecular standards (e.g., internal standards andexternal standards) can be run with the samples, and a standard curveconstructed based on ions generated from those standards. Using such astandard curve, the relative abundance of a given ion can be convertedinto an absolute amount of the original molecule. In certain preferredembodiments, an internal standard is used to generate a standard curvefor calculating the quantity of inventive protein biomarkers describedherein. Numerous other methods for relating the presence or amount of anion to the presence or amount of the original molecule are well known tothose of ordinary skill in the art.

Once the levels of the biomarkers of interest have been determined forthe biological sample being analyzed, they are compared to the levels inone or more control samples or to at least one expression profile mapdescribed herein. Comparison of levels according to methods of thepresent invention is preferably performed after the levels obtained havebeen corrected for both differences in the amount of sample assayed andvariability in the quality of the sample used (e.g., amount of proteinextracted). Correction may be carried out using different methodswell-known in the art. For example, the protein concentration of asample may be standardized using photometric or spectrometric methods orgel electrophoresis (as already mentioned above) before the sample isanalyzed.

For a given set of inventive proteins, comparison of an expressionpattern obtained for a biological sample against an expression profilemap established as described herein may comprise a comparison of thenormalized levels on a biomarker-by-biomarker basis and/or comparison ofratios of levels within the set of protein markers. In addition, theprotein expression pattern obtained for the biological sample beinganalyzed, may be compared against each of the expression profile maps(e.g., expression profile map for low or no risk of IPS, expressionprofile map for high risk of IPS, expression profile map for progressionof IPS, expression profile map for IPS responsive to treatment by aTNF-α inhibitor) or against an expression profile that definesdelineations made based upon all the IPS expression profile maps.

In another aspect of the invention, skilled physicians may select andprescribe treatments adapted to each individual subject based on themethods described herein of characterizing the risk a subject willdevelop IPS and/or a specific IPS sub-type following allogeneichematopoietic stem cell transplantation. In particular, the presentinvention provides physicians with a non-subjective means tocharacterize the risk a subject will develop IPS as early as day 0 posttissue transplant, which will allow for early treatment, whenintervention is likely to have its greatest effect. Selection of anappropriate therapeutic regimen for a given patient may be made basedsolely on the characterization of risk (e.g., a high level of risk)provided by the inventive methods. Alternatively, the physician may alsoconsider other clinical or pathological parameters used in existingmethods to diagnose IPS and assess its advancement.

In some aspects, GVHD, and IPS diagnosed by the methods of the presentinvention, may be treated with a TNF-α inhibitor. Recent studies haveshown that the lung fluid of patients with IPS contains higher thannormal amounts of inflammatory protein that may directly damage the lung(Yanik et al. Blood (2008) 112(8):3073-3081). One such example is tumornecrosis factor-alpha (TNF-α) which may also be involved in lung injuryfrom IPS. Tumor necrosis factor promotes the inflammatory response,which, in turn, causes many of the clinical problems associated withautoimmune diseases and/or alloimmune diseases, such as rheumatoidarthritis, ankylosing spondylitis, Crohn's disease, psoriasis andrefractory asthma.

Autoimmune diseases and/or alloimmune diseases are sometimes treated byusing a TNF-α inhibitor. For example, it has been shown that the TNF-αinhibitor etanercept can be used for the treatment of IPS (Yanik andCooke (2009) Blood. 113: 2869). In general, TNF-α inhibition can beachieved with a monoclonal antibody such as infliximab or adalimumab, orwith a circulating soluble receptor fusion protein such as etanercept.While most clinically useful TNF-α inhibitors are monoclonal antibodies,some are simple molecules, such as pentoxifylline and bupropion.Certolizumab pegol is a monoclonal antibody directed against tumornecrosis factor alpha. More precisely, it is a PEGylated Fab′ fragmentof a humanized TNF-α inhibitor monoclonal antibody.

It is further contemplated by the present invention that once a subjectis characterized as having an elevated risk of idiopathic pneumoniasyndrome (IPS) following allogeneic hematopoietic stem celltransplantation using the methods described above, a TNF-α inhibitor maybe administered to treat the subject. In one particular example, asubject can be administered a TNF-α inhibitor when levels of at leastone protein consisting of LPS binding protein, Mannose-bindingprotein-C, PGLYRP2, attractin and lumican are elevated prior to or postallogeneic hematopoietic stem cell transplant. In another example, asubject can be administered a TNF-α inhibitor when levels of at leastone protein consisting of Mannose-binding protein-C, PGLYRP2, attractinand lumican are elevated prior to or post allogeneic hematopoietic stemcell transplant.

Therefore, in another aspect of the invention, a TNF-α inhibitor (e.g.,etanercept), may be used to treat IPS in the subject who has beencharacterized with a sub-type of IPS that is responsive to treatment bya TNF-α inhibitor. The TNF-α inhibitor administered to the subject caninclude, but is not limited to, etanercept, infliximab, adalimumab,certolizumab pegol, golimumab (simponi), lenercept, semapimod (a mapKinhibitor that reduces production of TNFa), pentoxifylline, thalidomide,and benzopyranes.

In another aspect, the present invention provides a method for thetreatment and/or prevention of IPS. The method includes administering toa subject an effective amount of a compound that modulates the level ofat least one inventive biomarker described herein (e.g., an agent thatmodulates the level of the proteins listed in Table 1, 2, 3 and 4). Thecompound may be known in the art to act as a modulator of the level ofat least one inventive biomarker.

For example, the levels of the proteins proteins LPS binding protein,Mannose-binding protein-C, PGLYRP2, attractin and lumican, which areresponsible for the regulation of the immune response, were identifiedby the inventors as significantly modulated in the plasma of subject'shaving IPS compared to a control (see Example below). Thus, LPS bindingprotein, Mannose-binding protein-C, PGLYRP2, attractin and lumican areappropriate molecular targets for the treatment of IPS. Therefore,agents which are effective in modulating LPS binding protein,Mannose-binding protein-C, PGLYRP2, attractin and/or lumican level in asubject (e.g., an anti-LPS binding protein or an anti-lumican antibody)are contemplated by the present invention.

Subjects that can receive a treatment according to the present inventioninclude individuals that have been diagnosed with IPS using conventionalmethods (e.g., radiological examination, clinical observations) as wellas individuals that have been diagnosed with IPS using diagnosticmethods provided herein. Suitable subjects may or may not havepreviously received traditional treatment for the condition.

A treatment according to the methods of the present invention mayconsist of a single dose or a plurality of doses over a period of time.An agent used in the present invention, or pharmaceutical compositionthereof, may also be released from a depot form per treatment. Theadministration may be carried out in any convenient manner such as byinjection (subcutaneous, intravenous, intramuscular, intraperitoneal, orthe like), oral administration, topical administration, rectaladministration, or sublingual administration.

Effective dosages and administration regimens can be readily determinedby good medical practice and the clinical condition of the individualsubject. The frequency of administration will depend on thepharmacokinetic parameters of the active ingredient(s) and the route ofadministration. The optimal pharmaceutical formulation can be determineddepending upon the route of administration and desired dosage. Suchformulations may influence the physical state, stability, rate of invivo release, and rate of in vivo clearance of the administeredcompounds.

Depending on the route of administration, a suitable dose may becalculated according to body weight, body surface area, or organ size.Optimization of the appropriate dosage can readily be made by thoseskilled in the art in light of pharmacokinetic data observed in humanclinical trials. The final dosage regimen will be determined by theattending physician, considering various factors which modify the actionof drugs, e.g., the drug's specific activity, the severity of the damageand the responsiveness of the patient, the age, condition, body weight,sex and diet of the patient, the severity of any present infection, timeof administration and other clinical factors.

The rapid progression to respiratory failure and the high mortality rateafter IPS onset, despite advances in critical care, emphasizes the needto characterize the efficacy of a TNF-α inhibitor in treating idiopathicpneumonia syndrome (IPS) in a subject following allogeneic hematopoieticstem cell transplantation. Therefore, a method of characterizing theefficacy of a TNF-α inhibitor is contemplated by the present invention.The method includes obtaining a biological sample from the subject anddetermining the level of at least one protein of interest in the sample.The method further includes comparing the measured level of the at leastone protein to a control value and characterizing TNF-α inhibitor asbeing more effective in treating IPS when administered to the subject ifthe level of the at least on protein determined is increased ordecreased compared to a control value.

In another aspect, the present invention provides kits comprisingmaterials useful for carrying out the methods according to the presentinvention. The characterization procedures described herein may beperformed by diagnostic laboratories, experimental laboratories, orpractitioners. The invention provides kits, which can be used in thesedifferent settings.

Materials and reagents for characterizing biological samples obtainedfrom a subject according to the inventive methods may be assembledtogether in a kit. In certain aspects, an inventive kit comprises atleast one reagent that specifically detects levels of one or moreinventive protein markers, and instructions for using the kit accordingto a method of the invention. Each kit may preferably include thereagent, which renders the procedure specific. Thus, fordetecting/quantifying a protein marker (or an analog or fragmentthereof), the reagent that specifically detects levels of the proteinmay be an antibody that specifically binds to the protein marker (oranalog or fragment thereof).

Depending on the procedure, the kit may further comprise one or more of,extraction buffer and/or reagents, amplification buffer and/or reagents,hybridization buffer and/or reagents, immunodetection buffer and/orreagents, labeling buffer and/or reagents, and detection means.Protocols for using these buffers and reagents for performing differentsteps of the procedure may be included in the kit.

The reagents may be supplied in a solid (e.g., lyophilized) or liquidform. The kits of the present invention may optionally comprisedifferent containers (e.g., vial, ampoule, test tube, flask or bottle)for each individual buffer and/or reagent. Each component will generallybe suitable as aliquoted in its respective container or provided in aconcentrated form. Other containers suitable for conducting certainsteps of the disclosed methods may also be provided. The individualcontainers of the kit are preferably maintained in close confinement forcommercial sale.

In certain aspects, the kits of the present invention further includecontrol samples. In other aspects of the invention, the inventive kitsinclude at least one expression profile map for a GVHD, IPS and/or IPSsub-type as described herein for use as comparison template. Preferably,the expression profile map is digital information stored in acomputer-readable medium.

Instructions for using the kit, according to one or more methods of theinvention, may comprise instructions for processing the biologicalsample obtained from the subject, and/or for performing the test,instructions for interpreting the results. As well as a notice in theform prescribed by a governmental agency (e.g., FDA) regulating themanufacture, use or sale of pharmaceuticals or biological products.

Example

Using label free expression proteomic and bioinformatic analyses, wehave discovered and verified novel biomarkers of IPS progression aftertransplant. In particular, we identified markers that can subtype stemcell transplant patients who: 1) are likely to develop IPS, and 2) arelikely to respond well to etanercept treatment. These markers can beused as prognostic tools to effectively target and trigger therapeuticintervention earlier than currently described. The following outlinesthe specific markers and assays in human plasma samples to detect thesemarkers by mass spectrometry, ELISA, or related antibody and othermethods.

Plasma EDTA was collected from IPS patients post blood marrow transplantand at the time of IPS diagnosis, while for the control (non-progressorsgroup) samples were post-transplant and 14 days after transplant. Thus,there were two patient types; bone marrow transplant /− IPS and bonemarrow transplant /+ IPS and two times of collection, post-BMT andapproximately 2 weeks later. A total of 22 samples were depleted of the7 most abundant proteins using standard methods. One hundred microgramsof each sample was digested with trypsin and a fraction of this digest(600 nanograms) was analyzed by LC/MS/MS via capillary liquidchromatography and a LTQ-FT. Automated differential quantification ofpeptides was accomplished using ProteoMarker. Peptide and proteinidentifications were integrated with these quantifications and used forstatistical analysis via mixed multi-way ANOVA. 2025 peptides across 22plasma samples were tracked in our analysis. Of these peptides, 1253received a statistically significant sequence assignment and of those552 peptides had p-values of <0.05 when analyzed by ANOVA (withadjustment for false discovery rates) for disease and time comparisonsof cases versus controls. These peptides are derived from 102 distinctproteins and changes in their abundance in a patient are significantpredictors of IPS progression. The 102 proteins identified along withtheir corresponding change in abundance (identified as + for increase inabundance and − for decrease in abundance) are listed in Table 5.

TABLE 5 Desc t A1BG Alpha-1B-glycoprotein −2.4717 A2MAlpha-2-macroglobulin −1.92582 AFM Afamin −3.74093 AGT Angiotensinogen4.317323 AHSG Alpha-2-HS-glycoprotein −4.69926 ALDOBFructose-bisphosphate aldolase B −2.0052 AMBP AMBP protein 0.341469 APCSSerum amyloid P-component 2.578971 APOA1 Apolipoprotein A-I −3.44706APOA2 Apolipoprotein A-II −3.44494 APOA4 Apolipoprotein A-IV 0.140954APOA4 apolipoprotein A-IV precursor −2.11612 APOB Apolipoprotein B-1001.123166 APOC3 Apolipoprotein C-III 1.616698 APOD Apolipoprotein D−2.462 APOE Apolipoprotein E 3.272737 APOF apolipoprotein F precursor−2.17694 APOH Beta-2-glycoprotein 1 −2.92375 APOL1 Isoform 2 ofApolipoprotein-L1 −1.59928 APOM Apolipoprotein M −4.99022 ATRN Isoform 3of Attractin −2.13266 AZGP1 alpha-2-glycoprotein 1, zinc 2.018238 BTDbiotinidase precursor −2.56362 C14orf39 Protein SIX6OS1 3.640615 C1QBcomplement component 1, q subcomponent, −1.97171 B chain precursor C1QCComplement C1q subcomponent subunit C 3.220511 C1R Complement C1rsubcomponent −0.65396 C1RL Complement C1r subcomponent-like protein−2.2814 C1S Uncharacterized Protein 0.860878 C3 Complement C3 (Fragment)−1.30768 C4A Complement component 4A −1.97228 C4B complement component4B preproprotein 1.571717 C4BPA C4b-binding protein alpha chain 0.878785C4BPB Isoform 2 of C4b-binding protein beta chain −2.42133 C5 ComplementC5 −1.35444 C6 Complement Component Precursor −2.25676 C7 Complementcomponent C7 −2.01065 C8A Complement component C8 alpha chain −2.1207C8B Complement component C8 beta chain −2.0818 C8G Complement componentC8 gamma chain −1.95742 C9 Complement component C9 2.086113Carboxypeptidase N subunit −2.32317 CFB Isoform 1 of Complement factor B(Fragment) 1.979763 CFH Isoform 1 of Complement factor H −1.36525 CFHR2Isoform Short of Complement factor −2.1821 H-related protein 2 CFIComplement factor I −2.00425 CLEC3B Putative uncharacterized protein−2.19537 DKFZp686H17246 CP Ceruloplasmin 1.829568 CPB2 Isoform 1 ofCarboxypeptidase B2 −2.25166 CPN1 Carboxypeptidase N catalytic chain−2.19879 CRP Isoform 1 of C-reactive protein 5.245925 F12 CoagulationFactor −2.18843 F2 Prothrombin (Fragment) −1.39156 FCN3 Isoform 1 ofFicolin-3 1.856238 FGA Isoform 2 of Fibrinogen alpha chain 0.612848 GCvitamin D-binding protein precursor −2.1762 GMPR2 GMPR2 protein 2.702804GPX3 Glutathione peroxidase 3 2.028996 GSN Isoform 1 of Gelsolin−2.64335 HBB Hemoglobin subunit beta −2.12803 HPX Hemopexin −2.95159 HRGHistidine-rich glycoprotein −3.49181 IGFALS Insulin-like growthfactor-binding protein −2.15608 complex acid labile chain ITIH1Inter-alpha-trypsin inhibitor heavy chain H1 −3.19335 ITIH2Inter-alpha-trypsin inhibitor heavy chain H2 −3.99365 ITIH3 Isoform 1 ofInter-alpha-trypsin inhibitor −0.08417 heavy chain H3 KLKB1 PlasmaKallikrein Precursor −1.77692 KNG1 Isoform LMW of Kininogen-1 0.824532KRT1 Keratin, type II cytoskeletal 1 2.690668 LBPLipopolysaccharide-binding protein 3.048725 LOC653879 similar tocomplement component 3 −2.10055 LRG1 Leucine-rich alpha-2-glycoprotein3.361271 LUM Lumican −2.23514 MBL2 Mannose-binding protein C −1.97585ORM1 Alpha-1-acid glycoprotein 1 3.804263 ORM2 Alpha-1-acid glycoprotein2 2.96858 PGLYRP2 Isoform 1 of N-acetylmuramoyl-L-alanine −2.68326amidase PLG Plasminogen −2.23116 PON1 Serum paraoxonase/arylesterase 1−2.03837 PON3 Serum paraoxonase/lactonase 3 −1.90156 PROS1 VitaminK-dependent protein S −2.28215 RBP4 Retinol binding protein 4, plasma−0.03233 S100A9 Protein S100-A9 −2.76747 SAA1; SAA2 Serum amyloid Aprotein 2.706316 SAA1; SAA2 serum amyloid A2 isoform a 5.079694 SAA4Serum amyloid A-4 protein −1.70284 SERPINA1 Isoform 1 ofAlpha-1-antitrypsin 2.025056 SERPINA3 Isoform 1 ofAlpha-1-antichymotrypsin 3.737104 SERPINA4 Kallistatin −1.94552 SERPINA6Corticosteroid-binding globulin −2.90036 SERPINA7 Thyroxine-bindingglobulin −1.76982 SERPIND1 Serpin peptidase inhibitor, clade D −0.9005(Heparin cofactor), member 1 SERPINF1 Pigment epithelium-derived factor0.644739 SERPINF2 SERPINF2 protein −0.96973 SERPING1 Plasma protease C1inhibitor 1.318974 SHBG Isoform 1 of Sex hormone-binding globulin−2.19629 SLC26A6 Anchor protein 2.696059 SLC27A6 Long-chain fatty acidtransport protein 6 −1.78318 TCP11L1 cDNA FLJ11386 fis, cloneHEMBA1000523, −1.80354 weakly similar to TESTIS-SPECIFIC PROTEIN PBS13TTR Transthyretin −1.76341 UBTF Isoform UBF2 of Nucleolar transcription−3.74554 factor 1 VTN Vitronectin −3.16218

Table 5 represents 102 distinct proteins that were identified from thesignificant peptides. These represent proteins for which antibody assayscan be developed for clinical detection. These data was subsequentlyincorporated with existing molecular networks and disease specificliterature via Pathway Studio, IPA software packages and additionaltools for an extended biological analysis. Moreover, many proteinsidentified as significant in the label free analysis are consistent withstudies conducted on animal models of IPS. One example is lipidpolysaccharide binding protein (LBP). This protein is involved in theacute-phase immunologic response to gram-negative bacterial infectionsby binding to endogenous endotoxin (LPS), which is a potent enhancer ofinflammatory cytokine release. LPS levels rise in mice with IPS andstudies in experimental models suggest that LPS elicits a severe, acuteinflammatory response in the lungs (Cooke et al., Blood (1996) 88(8);3230-3239; Nelson et al., J. Infect Dis (1989) 159(2); 189-194; Smith etal., Am J Respir Cell Mol Biol (1998) 19(6):881-891.) In addition,proteins responsible for regulation of the immune response such aslumican and attractin (Carlson et al., J Biol Chem (2005)280(27):25541-25547; Duke-Cohan et al., Proc Natl Acad Sci USA (1998)95(19):11336-11341; Johnson et al., Invest Ophthalmol Vis Sci (2005)46(2):589-595; Wrenger et al., J Leukoc boil (2006) 80(3):621-629),which were previously not associated with this disease were identifiedas significant in the label free expression study. Lumican is ofparticular interest as it has been suggested that lumican regulatesantigen sensing by Toll-like receptors 4 and innate immune response.

To probe the potential for lumican abundance levels to be used as apotential early indicator for progression to IPS, we examined theindividual expression values for this protein in individual patients.This analysis has detected increased lumican levels prior to clinicaldiagnosis of this complication (day 0), the levels are most strikinglyelevated for the IPS patients that responded well to the specifictreatment strategy with etanercept. It is clear that therapy withetanercept can be guided by a molecular diagnostic in plasma forlumican, where bone-marrow transplant patients with elevated lumicanlevels would be treated immediately, with likely beneficial effects thatmight entirely suppress development of IPS.

In addition, a disease specific molecular network was generated viaincorporation of statistically significant proteins, existing molecularnetworks and disease specific literature. The data demonstrated thatattributes of the innate immune system contribute to thepatho-physiology of IPS in SCT patients and many proteins identified assignificant in the label free analysis are consistent with studiesconducted on animal models of this disease such as lipid polysaccharidebinding protein (LBP). In addition, proteins responsible for regulationof the immune response which were previously not associated with thisdisease were identified as significant in the label free expressionstudy. Finally, the observed increases of LBP in patients with IPS havebeen verified via ELISA techniques.

Experimental Procedures Patients and Patient Controls

Patients were recruited from May 2001 to February 2004 at the Blood andMarrow Transplantation Programs in the University of Michigan MedicalCenter and the Dana Faber Cancer Institute and were required to be atleast 1 year of age, were within 100 days of receiving an allogeneic SCTand had IPS as defined by the NIH working group criteria. Patients wereexcluded from this study after the time of enrollment if they: 1) had apositive culture of bronchoalvelor lavage fluid (BAL) for activepulmonary infection; 2) had hypotension in which inotropic support otherthan dopamine at less than 5 μg/kg per minute was required; 3) hadbacteremia within 48 hours before the entry into the study; 4) werepositive for cytomegalovirus viremia via CMV polymerase chain reactionor pp 65 antigenemia tests; 5) had systemic fungal or other nonbacterial infections; or 6) had clinical evidence for cardiacdysfunction as the cause of respiratory failure. Controls were from SCTpatients who had no complications from transplantation through day 10.

Diagnostic Procedures

Chest radiography, bronchoscopy with BAL, CMV blood assay, aerobic bloodcultures and clinical assessment of pulmonary dysfunction was performedon all patients at the time of study entry. Pulmonary function wasdetermined via blood gas measurements and chest radiographs wereperformed twice with the first 7 days of treatment and then weeklythrough day 28. CMV assays were performed weekly through day 28.

Plasma Samples

Blood was obtained for label free expression at the time of bone marrowtransplant and at either the time of IPS diagnosis for patients orobtained between 14 and 21 days after transplant for patient controls.Blood was collected in heparinzed tubes and plasma was separated andstored at −80° C. until analysis.

Experimental Design for Label Free Analysis

The categorical factors under study consist of a Disease Factor (DF)crossed with a Time factor (TF) for which their main effects andpossible interaction are of interest. The two levels of the Diseasefactor were the two groups of patients who both received the bone marrowtransplant (SCT) and who were further either diagnosed with IPS(Progressors—P) or underwent no complications (Controls—C). The twolevels of the time factor were the two time-points on the day of BMT(Day-0) and on the day of IPS diagnosis for both controls and patient(Day-X for Progressors and Day-14 for Controls). These later two days(Day-X or Day-14) were further assumed to be the same in the statisticalanalysis. The experimental units under study are the patients (samplesize: n=11), assumed to be independent and randomly sampled from theentire population meeting inclusion criteria. The Disease factorrepresents the variable over which repeated measures were made withineach experimental unit.

Further, each combination of Disease×Time treatment was randomized(without blocking) among 5 and 6 experimental units (biologicalreplicates) of Progressors and Controls respectively. Therefore, this isa Factorial Arrangement of treatments (Disease×Time) laid out on anunbalanced Completely Randomized Design (CRD) with repeated measures onone treatment (Disease).

Sample Preparation

Individual plasma samples were depleted of the seven most abundantproteins using a 4.6×100 mm multiple affinity removal system (MARS Hu7,Agilent Technologies, Santa Clara, Calif.) according to manufacturer'sinstructions. Each depleted sample was concentrated (5,000 molecularweight cutoff, Millipore, Billerica, Mass.) and buffer exchanged with 50mM Tris to a final volume of approximately 100 microliters. Totalprotein concentrations were determined by 2D Quant Kit as described bythe manufacturer (GE Healthcare Piscataway, N.J.) and 10 micorgrams offlow through and bound fractions for each sample were loaded onto a onedimensional SDS-PAGE gel (4-20% Tris-HCL) as a quality control measurefor the depletion step. Subsequent to digestion, each sample wasadjusted to 60 micrograms in 50 μL. Twenty microliters of 0.2% Rapigest(Waters, Milford, Mass.) and dithiotheritol to a final concentration of5 mM was added. The samples were reduced at 80° C. for 15 minutes andcooled to room temperature prior to alkylation with iodacetamide at afinal concentration of 10 mM for 30 minutes. Proteolytic digestion wasperformed with bovine trypsin (Promega, Madison, Wis.) with a finalenzyme to protein ration 1:10 (w/w) of for 18 hours at 37° C.

Label Free Expression Liquid Chromatography and Mass Spectrometry

Three hundred nanograms of each sample were analyzed by LC/MS/MS and theorder of sample injections randomized over all samples. Separation ofpeptides via capillary liquid chromatography was performed using aDionex Ultimate 3000 capillary LC system (Dionex Sunnyvale, Calif.).Mobile phase A (aqueous) contains 0.1% formic acid in 5% acetonitrileand mobile phase B (organic) contained 0.1% formic acid in 85%acetonitrile. Samples were trapped and desalted on-line in mobile phaseA at 10 μL/min for 10 minutes using a Dionex PepMap 100, (300 μm×5 mm)The sample was subsequently loaded onto a Dionex C18 PepMap (75 μm×15cm) reversed phase column with 5% mobile phase B. Separation wasobtained by employing a gradient of 6% to 28% mobile B at 0.300 μL/minover 100 minutes. The column was washed at 99% mobile phase B for 10minutes, followed by a re-equilibration at 100% A for 17 minutes. Massspectrometry analyses of samples were performed using a hybrid linearion trap Fourier-transformation cyclotron resonance mass spectrometer(LTQ-FT, Thermo, Waltham, Mass.). Positive mode electrospray wasconducted using a nanospray source and the mass spectrometer wasoperated at a resolution of 25,000. Quantitative and qualitative datawere acquired using alternating full MS scan and MS/MS scans in normalmode. Survey data were acquired from m/z of 400 to 1600 and up to 3precursors based on intensity, were interrogated by MS/MS per switch.Two micro scans were acquired for every precursor interrogated and MS/MSwas acquired as centroid data. The FT and LTQ were mass calibratedimmediately before the analysis using the instrument protocol. RawLC/MS/MS data was processed via Proteomarker software (Infochromics,Toronto, Canada).

Data Processing—Qualitative and Quantitative

The raw data were for each run were first extracted to provide MS/MSpeak lists for identification and intensity based profile peak lists forquantification. The MS/MS peak lists were subsequently searched byMascot version 2.2.0 (Matrix Science London, UK). The database used wasthe human International Protein Index (IPI) (68020 sequences). Searchsettings were as follows: trypsin enzyme specificity, mass accuracywindow for precursor ion, 10 ppm; mass accuracy window for fragmentions, 0.8 Daltons; variable modification, including onlycarbamidomethylation of cysteines and oxidation of methionine. Thecriteria for peptide identification were a mass accuracy of <10 ppm andan expectation value of p≦0.05. Proteins that had >2 peptides matchingthe above criteria were considered confirmed assignments while proteinsidentified with one peptide regardless of the Mascot score werehighlighted as tentative assignments.

Network Analysis of Label Free Expression Data

Following statistical analysis, significant proteins with theircorresponding abundance change were imported into Ingenuity PathwayAnalysis (Ingenuity Systems, Redwood City, Calif., USA). This softwareutilizes biomedical literature and existing protein interactiondatabases to elucidate biological networks within uploaded protein lists(Ingenuity®, www.ingenuity.com). This is accomplished by estimates ofsignificance and ranking of biological networks and pathways identifiedvia the right tailed distribution of Fisher's exact test. In addition,we have controlled the false discovery rate (FDR) by setting a thresholdin the software of q-values of 0.1, which means that no more than 10% ofall pathways identified as significant are false positives.

Results Label Free Expression Analysis

The sample depletion protocol was reproducible across individual samplesand yielded sufficient protein concentrations for the bound fractionwith ranges of 1.7 μg/μL to 5.4 μg/μL. Reproducible protein patterns via1D-SDS PAGE were observed across all samples for both flow-through andbound fractions (data not shown). These depleted samples weresubsequently digested and analyzed by LC/MS/MS as described in themethods section. The optimized LC/MS/MS analysis provided excellentchromatographic reproducibility across experimental groups andinjections.

Network Analysis of Significantly Changing Proteins

Proteins that were identified as significant in the above statisticalanalysis were subsequently imported into IPA for network analysis andtwo immune related networks were identified. FIG. 6 highlights thebiological functions associated with these networks and theirstatistical significance. Interestingly, the top functions identifiedwere immune related and were highly significant as 43 of the 79 proteinsimported into IPA were associated with immune related processes. Onecanonical pathway which was identified as significant was acute phaseresponse signaling which was anticipated as IPS elicits a severeinflammatory response in patients.

Biological Significance of Networks Identified Footprints of TNF-α andIL-6 Signaling

The acute phase response (APR) is an innate systemic immune response toinjury and/or infection. It involves the altered expression of proteinssynthesized in the liver whose plasma concentrations increase (positiveacute phase reactants) or decrease (negative acute phase reactants) inresponse to circulating inflammatory cytokines such IL-6, IL-1 andTNF-α. Due to this relationship, these serum proteins can serve as“readouts” of the Acute Phase Signaling Response (APSR) and the keycytokines involved in its regulation. Good correlation was observed forboth negative and positive acute phase reactants with respect to theirabundance change in plasma. Six of the eight negative acute phasereactants identified as significant in the interaction effect wereobserved to decrease in abundance while 16 of 28 positive acute phasereactants plasma abundances increased. This data provides additionalevidence and cross validation of cytokine up-regulation in IPS patientsas previous ELISA assays for TNF-α and IL-6 have shown significantincreases in protein concentrations in both BAL fluid and plasma of IPSpatients when compared to allogeneic HCT patients without IPS.

Activation of the Innate Immune Response

The innate immune response is primarily mediated by antigen presentingcells (APCs) and phagocytic cells both of which require activation viapattern recognition of evolutionary conserved structures on pathogens.These structures are also known as pathogen-associated molecularpatterns (PAMPs) and are detected via pattern recognition molecules suchas Toll like receptors (TLRs) and LPS binding protein (LBP). Oncedetected, their engagement with TLRs on the cell surface triggers acascade of signaling events, which culminate in the production ofproinflammatory cytokines. LBP is a well characterized acute phaseprotein which binds endotoxin. TLR4 activation and sensitivity toendotoxin relies on a series of interactions starting with LBP thatenables optimal presentation of endotoxin to TLR4. In addition tobacterial recognition, LBP also can indirectly regulate the inflammatoryresponse. Numerous studies in LBP knockout mice have shown that theabsence of LBP leads to reduce LPS responsiveness and immune activation.In addition, recent work have found acute phase LBP levels block TLR4signally and subsequent TNF-α secretion suggesting an inhibitory rolefor LBP at very high concentrations. In our study, average LBP levelswere slightly higher in IPS patients at day 0 than controls and asignificant increase in IPS patients was observed at diagnosis whencompared to controls. Moreover, this increase was observed in both thelabel free experiment as well as an ELISA detecting LBP on a largersubset from this cohort providing cross validation of the analyticalplatforms.

As mentioned above, aside from LBP a number of other pattern recognitionmolecules were identified as significant from our analysis. Mannosebinding protein C (MBL), peptidoglycan recognition protein 2 and lumicanare also pattern recognition proteins and had similar interactionprofiles with increases in abundance in IPS patients at day 0 whencompared to controls at the same timepoint. MBL is a serum protein,which is synthesized in the liver. It belongs to the collectin proteinfamily that consists of collagenous and lectin domains. In addition, itcontains a carbohydrate recognition domain, which enables it to bindpolysaccharide structures presented by pathogens and activatescomplement via the lectin pathway. Traditionally, its role in the innateimmune response and inflammation was thought to be attributed to itsability to trigger the lectin pathway but recent studies havedemonstrated an ability to modulate the inflammatory response. Theproposed mechanism by which this modulation occurs is via MBL acting asa TLR2/TLR6 co-receptor within the cell. In vitro studies have shownthat MBL binds to LTA and complexes with TLR 2 within the phagosomeincreasing efficiency of ligand delivery within the macrophage andsubsequent cytokine signaling. In our analysis, a single peptide wasidentified for MBL, therefore, it is not included in our confirmedprotein list. However, the MBL peptide had a very high mascot score(score=95) which corresponded to a 1.0e⁻⁰⁰⁹ probability of a correctassignment and was also significant for the interaction effect.

Whereas LBP and MBL have defined biological functions and wellestablished roles in the innate immune response, PGLYRP2 and lumicanhave recently been identified as pattern recognition proteins.Peptidoglycan recognition proteins (PGRPs) are innate immunity proteinsthat contain a well conserved peptidoglycan binding type 2 amidasedomain across most animals. In mammals, PGLYRP2 is a member of a familyof peptidoglycan recognition proteins that also includes PGLYRP1, 3 and4. Mammalian PGLYRP2 is a serum protein produced by the liver which haspeptidoglycan amidase acitivity and whose role in the immune responsewas proposed as an anti-inflammatory scavenger of peptidoglycan.However, recent studies in a peptidoglycan induced arthritis animalmodel have demonstrated that PGLYRP2 has pro-inflammatory properties. Inthis work, PGLYRP2 was found to locally modulate the inflammatoryresponse via cooperation with other pattern recognition moleculesspecifically Nod2 and TLR4 both of which are important regulators ofsignaling cascades involved in the production of pro-inflammatorycytokines. Five peptides of PGLYRP2 were identified in the label freeanalysis as significant for the interaction effect. The interactionprofile was similar to MBL with higher levels observed at day 0 for IPSthan controls at the same time point (FIG. 1).

As mentioned above, lumican is another protein identified in ouranalysis that has recently been shown to be involved in the innateimmune response and pattern recognition. Lumican is a small leucine richglycoprotein which is expressed is a variety of tissues and isextracellular matrix protein. A study which utilized a lumican knock outmodel to investigate the role of lumican in wound healing foundLum^(−/−) mice had poor recruitment of macrophages to the site of injuryas well as a significant decrease in the induction of inflammatorycytokines. Moreover, additional knock out studies have found functionalimpairment of the innate immune inflammatory response. In this work,Lum^(−/−) mice challenged with LPS were resistant to septic shock anddeath with poor induction of TNF-α and IL-6. Moreover this study foundlumican expression is induced during the innate immune response, LPSbinds to lumican and co-precipates with CD14 which is a key regulator ofLPS sensing. Taken together, this data suggest lumican may have animportant role in mediating antigen sensing and may enhance hostsensitivity to LPS. In the label free analysis, four peptides forlumican were identified as significant for the interaction effect. Threeof the five IPS patients had higher levels at day 0 for IPS thancontrols at the same time point. FIG. 2 highlights the distribution ofaverage peptide intensity for this protein. A 40% increase in averagepeptide abundance (non log scale) was observed for these three subjectswhen compared to other subjects analyzed in this study. Interestingly,these three IPS subjects responded to etanercept therapy with greaterthan a 100 day survival after treatment.

FIG. 3 highlights the distribution of average peptide intensity forlumican for 21 of the 22 patients. The data for patient number 46 (i.e.,the 22^(nd) patient) was omitted because of substantially elevatedcytokine levels were detected in this patient by ELISA. Lumican levelswere found to be down about 8% in progressors vs. non-proegrssors (day 0post transplantation compared to day 14 post transplantation) and up188% for responders vs. non responders to therapy (day 0 posttransplantation).

FIG. 4 highlights the distribution of average peptide intensity for MBL2Mannose-binding protein C for 21 of the 22 patients. MBL2Mannose-binding protein C levels were found to be up 1345% inprogressors vs. non progressors (day 0 post transplantation compared today 14 post transplantation) and up 58% in responders vs. non responders(day 0 post transplantation).

FIG. 5 highlights the distribution of average peptide intensity forPGLYRP peptidoglycan recognition protein for 21 of the 22 patients.PGLYRP peptidoglycan recognition protein levels were found to be up 189%in progressors vs. non progressors (day 0 post transplantation comparedto day 14 post transplantation) and up 88% in responders vs. nonresponders (day 0 post transplantation).

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims. All patents, publications andreferences cited in the foregoing specification are herein incorporatedby reference in their entirety.

1-12. (canceled)
 13. A method for characterizing the risk a subject willdevelop idiopathic pneumonia syndrome (IPS) following allogeneichematopoietic stem cell transplantation, the method comprising:obtaining a biological sample from the subject, wherein the subject hasreceived the allogeneic hematopoietic stem cell transplantation;determining in the biological sample a level of at least one proteinselected from the group consisting of Alpha-1B-glycoprotein,Alpha-2-macroglobulin, Afamin, Angiotensinogen, Alpha-2-HS-glycoprotein,Fructose-biphosphate aldolase B, AMBP protein, Serum amyloidP-component, Apolipoprotein A-I, Apolipoprotein A-II, ApolipoproteinA-IV, Apolipoprotein precursor, Apolipoprotein B-100, ApolipoproteinC-III, Apolipoprotein D, Apolipoprotein E, Apolipoprotein F precursor,Beta-2-glycoprotein 1, Isoform 2 of Apolipoprotein-L1, Apolipoprotein M,Isoform 3 of Attractin, alpha-2-glycoprotein 1 (zinc binding),biotimidase precursor, Protein SIX6OS1, complement component 1 (qsubcomponent, B chain precursor), Complement C1q subcomponent subunit C,Complement C1r subcomponent, Complement C1r subcomponent-like protein,Complement C3 (Fragment), Complement component 4A, Complement component4B preprotein, C4b-binding protein alpha chain, Isoform 2 of C4b-bindingprotein beta chain, Complement C5, Complement component C7, Complementcomponent C8 alpha chain, Complement component C8 beta chain, Complementcomponent C8 gamma chain, Complement component C9, Isoform 1 ofComplement factor B (fragment), Isoform 1 of Complement factor H,Isoform Short of Complement factor H-related protein 2, Complementfactor 1, DKFZp686H17246, Ceruloplasmin, Isoform 1 of CarboxypeptidaseB2, Carboxypeptidase N catalytic chain, Isoform 1 of C-reactive protein,Prothrombin (fragment), Isoform 1 of Ficolin-3, Isoform 2 of Fibrinogenalpha chain, vitamin D-binding protein precursor, GMPR2 protein,Glutathione peroxidase 3, Isoform 1 of Gelsolin, Hemoglobin subunitbeta, Hemopexin, Histidine-rich glycoprotein, Insulin-like growthfactor-binding protein complex acid labile chain, Inter-alpha-trypsininhibitor heavy chain H1, Inter-alpha-trypsin inhibitor heavy chain H2,Isoform 1 of Inter-alpha-trypsin inhibitor heavy chain H3, Isoform LMWof Kininogen-1, Type II cytoskeletal 1 Keratin,Lipopolysaccharide-binding protein, LOC653879 similar to complementcomponent 3, Leucine-rich alpha-2-glycoprotein, Lumican, Mannose-bindingprotein C, Complement component C6 precursor, Carboxypeptidase N subunitC1S, COAGgulation factor XII, Plasma Kallikrein Precursor, Alpha-1-acidglycoprotein 1, Alpha-1-acid glycoprotein 2, Isoform 1 ofN-acetylmuramoyl-L-alanine amidase, Plasminogen, Serumparaoxonase/arylesterase 1, Serum paraxonase/lactonase 3, VitaminK-dependent protein S, Retinol binding protein 4 (plasma), ProteinS100-A9, Serum amyloid A protein, Serum amyloid A2 isoform a, Serumamyloid A-4 protein, Isoform 1 of Alpha-1-antitrypsin, Isoform 1 ofAlpha-1-antichymotrypsin, Kallistatin, Corticosteroid-binding globulin,Thyroxine-binding globulin, Serpin peptidase inhibitor (clade D (Heparincofactor), member 1), Pigment epithelium-derived factor, SERPINF2protein, Plasma protease C1 inhibitor, Isoform 1 of Sex hormone-bindingglobulin, Anchor protein, Long-Chain fatty acid transport protein 6,TCP11L1, Transthyretin, Isoform UBF2 of Nucleolar transcription factor1, Vitronectin, analogs and fragments thereof, comparing the measuredlevel of the at least one protein to a control value; and characterizinga subject as at greater risk of developing IPS if the level of at leastone protein determined is increased or decreased compared to the controlvalue.
 14. The method of claim 13, further comprising generating anexpression profile based on the determined level of at least twoproteins, comparing the expression profile to a control expressionprofile, and characterizing a subject as at greater risk of developingIPS if the expression profile compared the control expression profile issubstantially different.
 15. The method of claim 13, wherein an increaseor decrease of at least about 5% in the determined level of the at leaston protein compared to the control value characterizes the subject atgreater risk of developing IPS.
 16. The method of claim 13, wherein anincrease or decrease of at least about 20% in the determined level ofthat at least one protein compared to the control value characterizesthe subject at greater risk of developing IPS.
 17. The method of claim13, wherein the level of at least one protein selected from the groupconsisting of Vitronectin, Isoform 3 of Attractin, Isoform 1 of Sexhormone-binding globulin, Alpha-2-HS-glycoprotein, Apolipoprotein M,Kallistatin, Isoform 1 of Gelsolin, Afamin, Inter-alpha-trypsininhibitor heavy chain H2, Putative uncharacterized proteinDKFZp686H17246, Complement component C8 alpha chain, ApolipoproteinA-II, Histidine-rich glycoprotein, PGLYRP1, PGLYRP2 Isoform 1 ofN-acetylmuramoyl-L-alanine amidase, Inter-alpha-trypsin inhibitor heavychain H1, Isoform UBF2 of Nucleolar transcription factor 1,Beta-2-glycoprotein 1, Lumican, Apolipoprotein A-I,Alpha-1B-glycoprotein, Plasminogen, Serum amyloid A-4 protein,Apolipoprotein D, Hemopexin, Protein S100-A9, vitamin D-binding proteinprecursor, SERPINF2 protein, Serum paraoxonase/arylesterase 1, Serpinpeptidase inhibitor Glade D (Heparin cofactor) member 1, Complementcomponent 4A, Complement component C7, Complement C3 (Fragment),Prothrombin (Fragment), Complement C5, Isoform 1 of Complement factor H,Isoform 1 of Inter-alpha-trypsin inhibitor heavy chain H3,Apolipoprotein A-IV, Apolipoprotein B-100, IgGFc-binding protein,Isoform 2 of Fibrinogen alpha chain, Isoform LMW of Kininogen-1,Complement C1q subcomponent subunit C, Complement component C9,Thyroxine-binding globulin, Ceruloplasmin, Serum amyloid P-component,C4b-binding protein alpha chain, Coagulation factor X, Complement factorI, Glutathione peroxidase 3, Anchor protein, Complement C1rsubcomponent, Plasma protease C1 inhibitor, Keratin type I cytoskeletal10, AMBP protein, complement component 1 q subcomponent B chainprecursor, Serum amyloid A protein, Isoform 1 of Alpha-1-antitrypsin,clusterin isoform 1, Alpha-1-acid glycoprotein 2, Pigmentepithelium-derived factor, 45 kDa protein, Fructose-bisphosphatealdolase B, Apolipoprotein E, Leucine-rich alpha-2-glycoprotein,Lipopolysaccharide-binding protein, zinc alpha-2-glycoprotein 1, IsoformShort of Complement factor H-related protein 2, GMPR2 protein,Alpha-1-acid glycoprotein 1, serum amyloid A2 isoform a, ProteinSIX6OS1, Isoform 1 of Alpha-1-antichymotrypsin, Angiotensinogen, Isoform1 of C-reactive protein, Complement factor H-related protein 1, andKeratin type II cytoskeletal 1 is determined and compared to a controlvalue, wherein an increase or decrease in the level of one at least oneprotein compared to a control level is indicative of IPS.
 18. The methodof claim 13, wherein the level of at least one protein selected from thegroup consisting of Prothrombin (Fragment), Complement C5, Isoform 1 ofComplement factor H, Isoform 1 of Inter-alpha-trypsin inhibitor heavychain H3, Apolipoprotein A-IV, Apolipoprotein B-100, IgGFc-bindingprotein, Isoform 2 of Fibrinogen alpha chain, Isoform LMW ofKininogen-1, Complement C1q subcomponent subunit C, Complement componentC9, Thyroxine-binding globulin, Ceruloplasmin, Serum amyloidP-component, C4b-binding protein alpha chain, Coagulation factor X,Complement factor I, Glutathione peroxidase 3, Anchor protein,Complement C1r subcomponent, Plasma protease C1 inhibitor, Keratin typeI cytoskeletal 10, AMBP protein, complement component 1 q subcomponent Bchain precursor, Serum amyloid A protein, Isoform 1 ofAlpha-1-antitrypsin, clusterin isoform 1, Alpha-1-acid glycoprotein 2,Pigment epithelium-derived factor, 45 kDa protein, Fructose-bisphosphatealdolase B, Apolipoprotein E, Leucine-rich alpha-2-glycoprotein,Lipopolysaccharide-binding protein, zinc alpha-2-glycoprotein 1, IsoformShort of Complement factor H-related protein 2, PGLYRP1, GMPR2 protein,Alpha-1-acid glycoprotein 1, serum amyloid A2 isoform a, ProteinSIX6OS1, Isoform 1 of Alpha-1-antichymotrypsin, Angiotensinogen, Isoform1 of C-reactive protein, Complement factor H-related protein 1, andKeratin type II cytoskeletal 1 is determined and compared to a controlvalue, wherein an increase in the level of one at least one proteincompared to a control level is indicative of IPS.
 19. The method ofclaim 13, wherein the level of at least one protein selected from thegroup consisting of Vitronectin, Isoform 3 of Attractin, Isoform 1 ofSex hormone-binding globulin, Alpha-2-HS-glycoprotein, Apolipoprotein M,Kallistatin, Isoform 1 of Gelsolin, Afamin, Inter-alpha-trypsininhibitor heavy chain H2, Putative uncharacterized proteinDKFZp686H17246, Complement component C8 alpha chain, ApolipoproteinA-II, Histidine-rich glycoprotein, PGLYRP2 Isoform 1 ofN-acetylmuramoyl-L-alanine amidase, Inter-alpha-trypsin inhibitor heavychain H1, Isoform UBF2 of Nucleolar transcription factor 1,Beta-2-glycoprotein 1, Lumican, Apolipoprotein A-I,Alpha-1B-glycoprotein, Plasminogen, Serum amyloid A-4 protein,Apolipoprotein D, Hemopexin, Protein S100-A9, vitamin D-binding proteinprecursor, SERPINF2 protein, Serum paraoxonase/arylesterase 1, Serpinpeptidase inhibitor Glade D (Heparin cofactor) member 1, Complementcomponent 4A, Complement component C7, and Complement C3 (Fragment) isdetermined and compared to a control value, wherein a decrease in thelevel of one at least one protein compared to a control level isindicative of IPS.
 20. The method of claim 13, wherein the level of atleast one protein selected from the group consisting of LPS bindingprotein, Mannose-binding protein-C, PGLYRP2, attractin and lumican isdetermined and compared to a control value, wherein an increase ordecrease in the level of one at least one protein compared to a controllevel is indicative of IPS.
 21. The method of claim 13, wherein the IPSis a subtype of idiopathic pneumonia syndrome that is responsive totreatment by a TNF-α inhibitor.
 22. The method of claim 13, wherein thebiological sample comprises a sample of blood, plasma, serum, orbronchoalveolar lavage fluid.
 23. A method for characterizing theprogression of idiopathic pneumonia syndrome (IPS) in a subjectfollowing allogeneic hematopoietic stem cell transplantation, the methodcomprising: obtaining a biological sample from the subject, wherein thesubject has received the allogeneic hematopoietic stem celltransplantation; and determining in the biological sample a level of atleast one protein selected from the group consisting ofAlpha-1B-glycoprotein, Alpha-2-macroglobulin, Afamin, Angiotensinogen,Alpha-2-HS-glycoprotein, Fructose-biphosphate aldolase B, AMBP protein,Serum amyloid P-component, Apolipoprotein A-I, Apolipoprotein A-II,Apolipoprotein A-IV, Apolipoprotein precursor, Apolipoprotein B-100,Apolipoprotein C-III, Apolipoprotein D, Apolipoprotein E, ApolipoproteinF precursor, Beta-2-glycoprotein 1, Isoform 2 of Apolipoprotein-L1,Apolipoprotein M, Isoform 3 of Attractin, alpha-2-glycoprotein 1 (zincbinding), biotimidase precursor, Protein SIX6OS1, complement component 1(q subcomponent, B chain precursor), Complement C1q subcomponent subunitC, Complement C1r subcomponent, Complement C1r subcomponent-likeprotein, Complement C3 (Fragment), Complement component 4A, Complementcomponent 4B preprotein, C4b-binding protein alpha chain, Isoform 2 ofC4b-binding protein beta chain, Complement C5, Complement component C7,Complement component C8 alpha chain, Complement component C8 beta chain,Complement component C8 gamma chain, Complement component C9, Isoform 1of Complement factor B (fragment), Isoform 1 of Complement factor H,Isoform Short of Complement factor H-related protein 2, Complementfactor 1, DKFZp686H17246, Ceruloplasmin, Isoform 1 of CarboxypeptidaseB2, Carboxypeptidase N catalytic chain, Isoform 1 of C-reactive protein,Prothrombin (fragment), Isoform 1 of Ficolin-3, Isoform 2 of Fibrinogenalpha chain, vitamin D-binding protein precursor, GMPR2 protein,Glutathione peroxidase 3, Isoform 1 of Gelsolin, Hemoglobin subunitbeta, Hemopexin, Histidine-rich glycoprotein, Insulin-like growthfactor-binding protein complex acid labile chain, Inter-alpha-trypsininhibitor heavy chain H1, Inter-alpha-trypsin inhibitor heavy chain H2,Isoform 1 of Inter-alpha-trypsin inhibitor heavy chain H3, Isoform LMWof Kininogen-1, Type II cytoskeletal 1 Keratin,Lipopolysaccharide-binding protein, LOC653879 similar to complementcomponent 3, Leucine-rich alpha-2-glycoprotein, Lumican, Mannose-bindingprotein C, Complement component C6 precursor, Carboxypeptidase N subunitC1S, COAGgulation factor XII, Plasma Kallikrein Precursor, Alpha-1-acidglycoprotein 1, Alpha-1-acid glycoprotein 2, Isoform 1 ofN-acetylmuramoyl-L-alanine amidase, Plasminogen, Serumparaoxonase/arylesterase 1, Serum paraxonase/lactonase 3, VitaminK-dependent protein S, Retinol binding protein 4 (plasma), ProteinS100-A9, Serum amyloid A protein, Serum amyloid A2 isoform a, Serumamyloid A-4 protein, Isoform 1 of Alpha-1-antitrypsin, Isoform 1 ofAlpha-1-antichymotrypsin, Kallistatin, Corticosteroid-binding globulin,Thyroxine-binding globulin, Serpin peptidase inhibitor (clade D (Heparincofactor), member 1), Pigment epithelium-derived factor, SERPINF2protein, Plasma protease C1 inhibitor, Isoform 1 of Sex hormone-bindingglobulin, Anchor protein, Long-Chain fatty acid transport protein 6,TCP11L1, Transthyretin, Isoform UBF2 of Nucleolar transcription factor1, Vitronectin, analogs and fragments thereof; comparing the measuredlevel of the at least one protein to a control value; and characterizingthe IPS as progressing if the level of at least one protein determinedis increased or decreased compared to the control value.
 24. The methodof claim 23, further comprising generating an expression profile basedon the determined level of at least two proteins, comparing theexpression profile to a control expression profile, and characterizingthe IPS in a subject as progressing if the expression profile comparedthe control expression profile is substantially different.
 25. Themethod of claim 23, wherein an increase or decrease of at least about 5%in the determined level of the at least on protein compared to thecontrol value characterizes the IPS in the subject as progressing. 26.The method of claim 23, wherein an increase or decrease of at leastabout 20% in the determined level of the at least on protein compared tothe control value characterizes the IPS in the subject as progressing.27. The method of claim 23, wherein the level of at least one proteinselected from the group consisting of Vitronectin, Isoform 3 ofAttractin, Isoform 1 of Sex hormone-binding globulin,Alpha-2-HS-glycoprotein, Apolipoprotein M, Kallistatin, Isoform 1 ofGelsolin, Afamin, Inter-alpha-trypsin inhibitor heavy chain H2, Putativeuncharacterized protein DKFZp686H17246, Complement component C8 alphachain, Apolipoprotein A-II, Histidine-rich glycoprotein, PGLYRP1,PGLYRP2 Isoform 1 of N-acetylmuramoyl-L-alanine amidase,Inter-alpha-trypsin inhibitor heavy chain H1, Isoform UBF2 of Nucleolartranscription factor 1, Beta-2-glycoprotein 1, Lumican, ApolipoproteinA-I, Alpha-1B-glycoprotein, Plasminogen, Serum amyloid A-4 protein,Apolipoprotein D, Hemopexin, Protein S100-A9, vitamin D-binding proteinprecursor, SERPINF2 protein, Serum paraoxonase/arylesterase 1, Serpinpeptidase inhibitor Glade D (Heparin cofactor) member 1, Complementcomponent 4A, Complement component C7, Complement C3 (Fragment),Prothrombin (Fragment), Complement C5, Isoform 1 of Complement factor H,Isoform 1 of Inter-alpha-trypsin inhibitor heavy chain H3,Apolipoprotein A-IV, Apolipoprotein B-100, IgGFc-binding protein,Isoform 2 of Fibrinogen alpha chain, Isoform LMW of Kininogen-1,Complement C1q subcomponent subunit C, Complement component C9,Thyroxine-binding globulin, Ceruloplasmin, Serum amyloid P-component,C4b-binding protein alpha chain, Coagulation factor X, Complement factorI, Glutathione peroxidase 3, Anchor protein, Complement C1rsubcomponent, Plasma protease C1 inhibitor, Keratin type I cytoskeletal10, AMBP protein, complement component 1 q subcomponent B chainprecursor, Serum amyloid A protein, Isoform 1 of Alpha-1-antitrypsin,clusterin isoform 1, Alpha-1-acid glycoprotein 2, Pigmentepithelium-derived factor, 45 kDa protein, Fructose-bisphosphatealdolase B, Apolipoprotein E, Leucine-rich alpha-2-glycoprotein,Lipopolysaccharide-binding protein, zinc alpha-2-glycoprotein 1, IsoformShort of Complement factor H-related protein 2, GMPR2 protein,Alpha-1-acid glycoprotein 1, serum amyloid A2 isoform a, ProteinSIX6OS1, Isoform 1 of Alpha-1-antichymotrypsin, Angiotensinogen, Isoform1 of C-reactive protein, Complement factor H-related protein 1, andKeratin type II cytoskeletal 1 is determined and compared to a controlvalue, wherein an increase or decrease in the level of one at least oneprotein compared to a control level is indicative of the IPS in thesubject progressing.
 28. The method of claim 23, wherein the level of atleast one protein selected from the group consisting of Prothrombin(Fragment), Complement C5, Isoform 1 of Complement factor H, Isoform 1of Inter-alpha-trypsin inhibitor heavy chain H3, Apolipoprotein A-IV,Apolipoprotein B-100, IgGFc-binding protein, Isoform 2 of Fibrinogenalpha chain, Isoform LMW of Kininogen-1, Complement C1q subcomponentsubunit C, Complement component C9, Thyroxine-binding globulin,Ceruloplasmin, Serum amyloid P-component, C4b-binding protein alphachain, Coagulation factor X, Complement factor I, Glutathione peroxidase3, Anchor protein, Complement C1r subcomponent, Plasma protease C1inhibitor, Keratin type I cytoskeletal 10, AMBP protein, complementcomponent 1 q subcomponent B chain precursor, Serum amyloid A protein,Isoform 1 of Alpha-1-antitrypsin, clusterin isoform 1, Alpha-1-acidglycoprotein 2, Pigment epithelium-derived factor, 45 kDa protein,Fructose-bisphosphate aldolase B, Apolipoprotein E, Leucine-richalpha-2-glycoprotein, Lipopolysaccharide-binding protein, zincalpha-2-glycoprotein 1, Isoform Short of Complement factor H-relatedprotein 2, PGLYRP1, GMPR2 protein, Alpha-1-acid glycoprotein 1, serumamyloid A2 isoform a, Protein SIX6OS1, Isoform 1 ofAlpha-1-antichymotrypsin, Angiotensinogen, Isoform 1 of C-reactiveprotein, Complement factor H-related protein 1, and Keratin type IIcytoskeletal 1 is determined and compared to a control value, wherein anincrease in the level of one at least one protein compared to a controllevel is indicative of the IPS in the subject progressing.
 29. Themethod of claim 23, wherein the level of at least one protein selectedfrom the group consisting of Vitronectin, Isoform 3 of Attractin,Isoform 1 of Sex hormone-binding globulin, Alpha-2-HS-glycoprotein,Apolipoprotein M, Kallistatin, Isoform 1 of Gelsolin, Afamin,Inter-alpha-trypsin inhibitor heavy chain H2, Putative uncharacterizedprotein DKFZp686H17246, Complement component C8 alpha chain,Apolipoprotein A-II, Histidine-rich glycoprotein, PGLYRP2 Isoform 1 ofN-acetylmuramoyl-L-alanine amidase, Inter-alpha-trypsin inhibitor heavychain H1, Isoform UBF2 of Nucleolar transcription factor 1,Beta-2-glycoprotein 1, Lumican, Apolipoprotein A-I,Alpha-1B-glycoprotein, Plasminogen, Serum amyloid A-4 protein,Apolipoprotein D, Hemopexin, Protein S100-A9, vitamin D-binding proteinprecursor, SERPINF2 protein, Serum paraoxonase/arylesterase 1, Serpinpeptidase inhibitor Glade D (Heparin cofactor) member 1, Complementcomponent 4A, Complement component C7, and Complement C3 (Fragment) isdetermined and compared to a control value, wherein a decrease in thelevel of one at least one protein compared to a control level isindicative of the IPS in the subject progressing.
 30. The method ofclaim 23, wherein the level of at least one protein selected from thegroup consisting of LPS binding protein, Mannose-binding protein-C,PGLYRP2, attractin and lumican is determined and compared to a controlvalue, wherein an increase or decrease in the level of one at least oneprotein compared to a control level is indicative of the IPS in thesubject progressing.
 31. The method of claim 23, wherein the IPS is asubtype of idiopathic pneumonia syndrome that is responsive to treatmentby a TNF-α inhibitor.
 32. The method of claim 23, wherein the biologicalsample comprises a sample of blood, plasma, serum, or bronchoalveolarlavage fluid. 33-51. (canceled)